Selective androgen receptor modulators and methods for their identification, design and use

ABSTRACT

Selective androgen receptor modulators (SARMs) having antagonist activity in hormone-dependent tumors while exhibiting no activity or agonist activity against other nontumor tissues containing the androgen receptor as well as methods for identifying, designing and using SARMs are provided.

INTRODUCTION

This application claims priority from U.S. Provisional Application Ser. No. 60/214,392, filed Jun. 28, 2000, from U.S. Provisional Application Ser. No. 60/233,519, filed Sep. 19, 2000, from U.S. Provisional Application Ser. No. 60/284,617, filed Apr. 18, 2001, from U.S. Provisional Application Ser. No. 60/284,438, filed Apr. 18, 2001, and from U.S. Provisional Application Ser. No. 60/284,730, filed Apr. 18, 2001, each of which are incorporated herein by reference in their entirety.

FIELD OF INVENTION

Selective androgen receptor modulators (SARMs) have now been identified which exhibit antagonistic activity against hormone-dependent tumors while exhibiting no activity or more preferably agonist activity against other nontumor tissues containing the androgen receptor. The present invention relates to methods for using these SARMs in the treatment of conditions remediable by administration of an androgen receptor modulator. The present invention also relates to methods for designing and identifying new SARMs that exhibit antagonistic activity against hormone-dependent tumors while exhibiting no activity, or more preferably agonist activity, against other nontumor tissues containing the androgen receptor. This invention also relates to structure coordinates of an androgen receptor ligand binding domain or ligand binding domain complex and method of using these structure coordinates for designing and selecting new SARMs that modulate androgen receptors.

BACKGROUND OF THE INVENTION

The androgen receptor (AR) is a member of the steroid nuclear-receptor superfamily of ligand-dependent transcription factors and is widely distributed among reproductive and nonreproductive tissues, including the prostate and seminal vesicles, male and female genitalia, skin, testis, ovary, cartilage, sebaceous glands, hair follicles, sweat glands, cardiac muscle, skeletal and smooth muscle, gastrointestinal vesicular cells, thyroid follicular cells, adrenal cortex, liver, pineal, and numerous brain cortical and subcortical regions, including spinal motor neurons (Negro-Vilar, A. JCE&M 1999 54(10):3459-62). As with the other members of the steroid receptor family, AR has several functional domains including a DNA binding domain (DBD), and a 261 residue ligand-binding domain (LBD) (Mw=30,245 Da) that contains the androgen binding site, and is responsible for switching on the androgen function. The cDNA and amino acid sequences of human and rat androgen receptors have been described (Proc. Natl. Acad. Sci. U.S.A. 1988 85: 7211-7215).

AR is an important target in multiple areas of drug discovery and patient therapy. In Oncology, for example, inhibitors (antagonists or partial antagonists) of the androgen receptor function are useful for the treatment of androgen dependent prostate cancer while agonists or partial agonists of the AR are applicable to the treatment of breast cancer. For metabolic and endocrine diseases disorders, for example, agonists or partial agonists of the androgen receptor function are useful for the treatment of age-related diseases and conditions of cachexia in several disease states including, but not limited to, AIDS. Functional AR has also been identified in various bone cells and androgen administration has beneficial effects on skeletal development and maintenance in men and women.

Progress of androgen therapy has been limited by the inability to separate desirable androgenic activities from undesirable or dose limiting side effects. However, recent advances in the development of selective estrogen receptor modulators (SERMs), with a great degree of tissue selectivity in targeting the estrogen receptor while eliminating undesired side effects, has resulted in the suggestion of SARMs, selective androgen receptor modulators (Negro-Vilar, A. JCE&M 1999 54(10):3459-62; Reid et al. Investigational New Drugs 1999 17:271-284).

U.S. Pat. No.6,017,924 discloses non-steroidal compounds characterized as high affinity, high specificity agonists, partial agonists (i.e. partial activators and/or tissue-specific activators) and antagonists for androgen receptors based upon a “cis-trans” or “co-transfection” assays. Non-steroidal compounds characterized as high affinity, high specificity agonists, partial agonists (i.e. partial activators and/or tissue-specific activators) and antagonists for androgen receptors via the “cis-trans” or “co-transfection” assays are also described in WO 01/16108, WO 01/16133, and WO 01/16139. This co-transfection assay (Evans et al. Science 1988 240:889-95) is suggested to provide a method for identifying functional agonists and partial agonists which mimic, or antagonists which inhibit, the effect of native hormones, and quantifying their activity for responsive intracellular receptor proteins.

In addition, hydroxyflutamide, a known AR antagonist in most tissues, has been suggested to function as a selective AR modulator (SARM) for effects on IL-6 production by osteoblasts (Hofbauer et al. J. Bone Miner. Res. 1999 14:1330-1337).

Hydroxyflutamide and Casodex, both known to be full AR antagonists in most tissues, have been shown, in AR-transfected PC3 cells, to activate MAP kinases Erk-1 and Erk-2 in an AR dependent fashion similar to DHT (Peterziel et. al. Oncogene 18, 6322-6329 (1999)).

The compound LGD2226, a non-steroidal AR agonist, has also been characterized as a selective androgen receptor modulator for use in the treatment of androgen-related diseases such as osteoporosis, male hormone replacement, male and female sexual dysfunction and cachexia (SCRIP—World Pharmaceutical New FILED 12 May 2000; WO 01/16108; WO 01/16133; and WO 01/16139).

The compound LG120907, a non-steroidal AR antagonist, has been shown, in rats, to have reduced antagonist effects on the hypothalamic axis and on libido (reproductive rate) as compared to other clinically used AR antagonists, such as Casodex. As such, LG120907 has been characterized as a selective androgen receptor modulator for the treatment of prostate cancer (Wang et. al. Poster#P3-126, Endocrine Society 80^(th) Annual Meeting (1998), Hamann et. al. Presentation#S39-2, Endocrine Society 80^(th) Annual Meeting (1998)).

Recent reports exploring the nogenotropic effects of sex steroid hormones such as DHT and E2 on the AR and ER, clearly show that both receptors regulate functions not specifically involved with transcriptional events (Kousteni et. all., Cell 104, 719-730 (2001)). The antiapoptic effects of ER and AR have been shown to be inducible by ligands that have no effects on transcription. It has also been shown that ligands that have effects on transcription can have no antiapoptotic effect.

SARMs exhibiting a difference-in-kind of the modulation effected in tumors containing the androgen receptor relative to the modulation effected in other, nontumor tissues containing the androgen receptor (especially, antagonist activity in tumors versus agonist activity in other, nonmalignant tissues containing the androgen receptor), have heretofore been neither disclosed nor suggested. The present invention provides SARMs, and methods for identifying and designing such SARMs, which exhibit antagonist activity in hormone-dependent tumors while exhibiting no activity, or more preferably agonist activity, against other nontumor tissues containing the androgen receptor. As described below, these SARMs can be employed, for example, to treat hormone-dependent tumors such as prostate cancer in patients by both inhibiting the growth of the tumor while mitigating side effects such as muscle wasting/cachexia, loss of libido, osteoporosis and gynecomastia. The term “patient” as used herein denotes an animal, preferably a mammal such as a dog, cat, or, most preferably, a human.

SUMMARY OF THE INVENTION

An object of the present invention is to provide methods for identifying SARMs having antagonist activity against hormone-dependent tumors while exhibiting no activity, or more preferably agonist activity, against other nontumor tissues containing the androgen receptor. In one embodiment, antagonist activity in hormone-dependent tumors is ascertained via screening for inhibition of growth, either in vitro or in vivo, in hormone-dependent tumor cell lines. In this embodiment, the activity of a potential SARM is also assessed in a normal, nontumor cell line. Alternatively, an animal model bearing a hormone-dependent tumor can be used to assess the antagonist activity of a potential SARM against the tumor as well as its activity in nontumor tissues in the animal.

Another object of the present invention is to provide methods for designing SARMs having antagonist activity in hormone-dependent tumors while exhibiting no activity, or more preferably agonist activity, against other nontumor tissues containing the androgen receptor by using information about the AR crystal structure and the estrogen receptor (ER) crystal structure with estradiol, tamoxifen or raloxifen.

Another object of the present invention is to provide molecule or molecular complexes comprising all or any part of a ligand binding site defined by structure coordinates of an androgen-receptor ligand binding domain (AR-LBD) amino acids V685, L700, L701, S702, S703, L704, N705, E706, L707, G708, E709, Q711, A735, I737, Q738, Y739, S740, W741, M742, G743, L744, M745, V746, F747, A748, M749, G750, R752, Y763, F764, A765, L768, F770, M780, M787, I869, L873, H874, F876, T877, F878, M894, M895, A896, E897, I898, I899, S900, V901, Q902, V903, P904, K905, I906 and L907 according to Table A as provided herein, or a mutant or homologue of said molecule or molecular complex for use in identifying SARMs.

Another object of the present invention is to provide machine-readable data storage media comprising a data storage material encoded with machine readable data, wherein the data is defined by the structure coordinates of an AR-LBD with an AR-LBD ligand or ligand complex according to Table A or a homologue of said complex, wherein said homologue comprises backbone atoms that have a root mean square deviation from the backbone atoms of the complex of not more than 3.0 Å.

Another object of the present invention is to provide a binding site in AR-LBD for an AR modulator in which a portion of said ligand is in van der Walls contact or hydrogen bonding contact with any portion or all of residues V685, L700, L701, S702, S703, L704, N705, E706, L707, G708, E709, Q711, A735, I737, Q738, Y739, S740, W741, M742, G743, L744, M745, V746, F747, A748, M749, G750, R752, Y763, F764, A765, L768, F770, M780, M787, I869, L873, H874, F876, T877, F878, L880, L881, V889, F891, P892, E893, M894, M895, A896, E897, I898, I899, S900, V901, Q902, V903, P904, K905, I906 or L907 of AR-LBD according to Table A. In a preferred embodiment, the binding site is a homologue or mutant with 25%-95% identity to residues V685, L700, L701, S702, S703, L704, N705, E706, L707, G708, E709, Q711, A735, I737, Q738, Y739, S740, W741, M742, G743, L744, M745, V746, F747, A748, M749, G750, R752, Y763, F764, A765, L768, F770, M780, M787, I869, L873, H874, F876, T877, F878, L880, L881, V889, F891, P892, E893, M894, M895, A896, E897, I898, I899, S900, V901, Q902, V903, P904, K905, I906 or L907 of AR-LBD according to Table A as provided herein.

Another object of the present invention is to provide SARMs having antagonist activity in hormone-dependent tumors while exhibiting no activity, or more preferably agonist activity, against other nontumor tissues containing the androgen receptor, as well as pharmaceutical compositions comprising at least one such SARM and a pharmaceutically acceptable carrier. In a preferred embodiment, the SARM is identified or designed in accordance with a method of the present invention.

Another object of the present invention is to provide a method for inhibiting the growth of hormone-dependent tumor cells comprising contacting the tumor cells with a SARM having antagonist activity in hormone-dependent tumors while exhibiting no activity, or more preferably agonist activity against other nontumor tissues containing the androgen receptor.

Unless otherwise indicated, SARMs of the present invention having antagonist activity against hormone-dependent tumors while exhibiting no activity, more preferably agonist activity against other nontumor tissues containing the androgen receptor are contemplated as further exhibiting, in all embodiments of the invention, agonist, antagonist or no activity against normal prostate tissue.

Yet another object of the present invention is to provide methods for using these SARMs in the treatment of conditions remediable by administration of an androgen receptor modulator as described herein including, but not limited to, hirsutism, acne, seborrhea, Alzheimer's disease, androgenic alopecia, hypogonadism, hyperpilosity, benign prostate hypertrophia, adenomas and neoplasias of the prostate (such as advanced metastatic prostate cancer), treatment of benign or malignant tumor cells containing the androgen receptor such as is the case for breast, brain, skin, ovarian, bladder, lymphatic, liver and kidney cancers, pancreatic cancers, modulation of VEGF expression and the applications therein for use as antiangiogenic agents, osteoporosis, suppressing spermatogenesis, libido, cachexia, endometriosis, polycystic ovary syndrome, anorexia, androgen dependent age-related diseases and conditions, such as androgen supplement for age-related decreased testosterone levels in men, male menopause, male hormone replacement, male and female sexual dysfunction, and inhibition of muscular atrophy in ambulatory patients. Particularly preferred, is the use of SARMs for the treatment of hormone-dependent tumors, particularly early stage prostate cancers, and for chemoprevention of hormone-dependent cancer, particularly prostate cancers.

All documents referred to herein, including but not limited to U.S. patent applications, are incorporated herein by reference in their entirety.

DETAILED DESCRIPTION OF THE INVENTION

Selective androgen receptor modulators (SARMs) have now been identified with antagonist activity against hormone-dependent tumors while exhibiting no activity, or more preferably agonist activity against other (i.e., one or more) nontumor tissues containing the androgen receptor. SARMs of the present invention exhibiting antagonist activity against hormone-dependent tumors and no activity against other nontumor tissues containing the androgen receptor may also be referred to as specific androgen receptor modulators.

For purposes of the present invention by “nontumor”, noncancerous” or “nonmalignant” androgen receptor containing tissues it is meant to include, but is not limited to, seminal vesicles, male and female genitalia, skin, testis, ovary, cartilage, sebaceous glands, hair follicles, sweat glands, muscle such as cardiac muscle, skeletal and smooth muscle, gastrointestinal vesicular cells, thyroid follicular cells, adrenal cortex, liver, pineal, bone, stromal cells, kidney tubules, urinary bladder and numerous brain cortical and subcortical regions, including spinal motor neurons.

Unless otherwise indicated, SARMs of the present invention having antagonist activity against hormone-dependent tumors while exhibiting no activity, or more preferably agonist activity, against other nontumor tissues containing the androgen receptor are contemplated as further exhibiting, in all. embodiments of the invention, agonist, antagonist or no activity against normal prostate tissue.

As used herein, the phrase “no activity or agonist activity” preferably denotes compounds with an activation effect (greater than 5%) in vivo as compared to control animals on the weights of the ventral prostate, seminal vesicles, levator ani and/or luteinizing hormone serum levels, and most preferably activity which maintains average normal bone density, average normal muscle mass, average normal reproductive function, and/or average normal libido seen in ugonadal warm-blooded male mammals, preferably human males. “No activity or agonist activity” of SARMS of the present invention is preferably exhibited at the same amount or range of amounts that exhibit antagonist activity against hormone-dependent tumors. When administered to a patient, this amount or range of amounts, wherein the SARM exhibits antagonist activity against hormone-dependent tumors while exhibiting no activity or more preferably agonist activity against other nontumor containing tissues, is the preferred therapeutically useful range. As will be understood by those of skill in the art upon reading this disclosure, SARMs of the present invention, when used in amounts outside the preferred therapeutically useful range, may exhibit the same antagonist activity against hormone-dependent tumors and no activity or agonist activity against nontumor containing tissues or may no longer exhibit the same specificity or selectivity. For example, SARMs of the present invention, when used in amounts exceeding the preferred therapeutically useful range may exhibit some antagonist activity in nontumor containing tissues.

The present invention relates to SARMs with these dual activities, methods for identifying these SARMs, pharmaceutical compositions comprising these SARMs, and methods of using these SARMs in the treatment of androgen receptor mediated diseases and disorders.

For example, SARMs of the present invention are useful in selectively inhibiting the growth of hormone-dependent tumors while preferably activating androgen receptor activity in other nontumor, meaning noncancerous or nonmalignant, androgen receptor containing tissues. Accordingly, the SARMs of the present invention are useful in treating tumors including, but not limited to, androgen receptor containing tumors such as prostate, breast, brain, skin, ovarian, bladder, lymphatic, liver and kidney cancers, and pancreatic cancers, while mitigating or eliminating unwanted side effects associated with inhibition of androgen receptor activity in other nontumor androgen receptor containing tissues. Some of the potential unwanted side effects which result from antagonizing the normal function of androgens such as dihydrotestosterone (DHT) seen, for example, with current antiandrogen therapy in the treatment of prostate cancer include, but are not limited to, muscle wasting/cachexia, loss of libido, osteoporosis and gynecomastia.

An additional preferred use of such SARMs is in the area of chemoprevention, particularly as it pertains to prostate cancer. SARMs of the present invention can be administered after radical prostectomy during the period of “watchfiul waiting” to decrease the incidence of reoccurrence of metastatic prostate cancer.

In addition, these SARMs are useful in the treatment of androgen dependent age-related diseases and conditions including, without limitation, cachexia and osteoporosis. Such agents provide an orally bioavailable androgen replacement therapy that does not suffer from the increased risk of prostate cancer seen with traditional androgen agonists.

The SARMs of the present invention are also expected to be useful in treating other conditions remediable by administration of an androgen receptor modulator such as hirsutism, acne, seborrhea, Alzheimer's disease, androgenic alopecia, hypogonadism, hyperpilosity, benign prostate hypertrophia, modulation of VEGF expression and the applications therein for use as antiangiogenic agents, suppressing spermatogenesis, libido, endometriosis, polycystic ovary syndrome, anorexia, and androgen dependent age-related diseases and conditions, such as androgen supplement for age-related decreased testosterone levels in men, male menopause, male hormone replacement, male and female sexual dysfunction, and inhibition of muscular atrophy in ambulatory patients.

Various methods for identifying SARMs having antagonist activity against hormone-dependent tumors while exhibiting no activity, or more preferably agonist activity against other nontumor tissues containing the androgen receptor can be used. In one embodiment, antagonist activity in hormone-dependent tumors is ascertained via screening for inhibition of growth, either in vitro or in vivo, in hormone-dependent tumor cell lines. Examples of hormone-dependent tumor cell lines which can be used for screening potential SARMs include, but are not limited to, human breast tumor cell line MDA MB453, human breast tumor cell line ZR-75-1, murine breast line Shionogi, rat prostate adenocarcinoma line Dunning R-3327, human prostate tumor cell line MDA PCa 2a and PCa 2b, human prostate cell line LNCap, human prostate tumor cell line CWR22, human prostate tumor cell line LuCaP 35 and LuCaP 23.12, human prostate cell line LAPC-4 and LAPC-9, human prostate tumor cell line PC-295, human prostate tumor cell line PC-310, and human osteosarcoma cell line MG-63. These experimental human and murine prostate and breast cell lines and the tumor model systems derived therein are well accepted by those of skill in the art as indicative of the pharmacology of human hormone-dependent tumors, such as prostate cancer. Examples of the relationship of such models to the human disease state can be found in, but are not limited to, the following references and the references contained therein, Jacques et. al. Endocrinology 140, 416-421 (1999); Yeap et. al. Endocrinology 140, 3282-3291 (1999), Sharma et. al. Oncogene 18, 5349-5355 (1999), Isaacs, J. T. Urol. Oncol. 2, 115-116 (1996), Bentei et. al. In Vitro Cell Dev. Biol. 35, 655-662 (1999), Suzuki et. al. J. Steroid Biochem. Mol. Biol. 37, 559-567 (1990), Peehl, D. M. Urol. Oncol. 2, 100-102 (1996), Wytske et. al. Urol. Oncol. 2, 122-125 (1996), Leland, C. W. K. Urol. Oncol. 2, 126-128 (1996), Buhler et. al. The Prostate 43, 63-70 (2000), Navone et. al. Clin. Cancer Res. 6, 1190-1197 (2000), Etreby et. al. The Prostate 42, 99-106 (2000), Jongsma et. al. Cancer Res. 60, 741-748 (2000), Jongsma et. al Amer. J. Path. 154, 543-551 (1999), Ye et. al Clin. Cancer Res. 5, 2171-2177 (1999), Navone et. al. Clin. Cancer Res. 3, 2493-2500 (1997), Klein et. al. Nature Medicine 3, 402-408 (1997), Chen et. al. Cancer Res. 58, 2777-2783 (1998), and Craft et. al. Cancer Res. 59, 5030-5036 (1999).

In this embodiment, the agonist or antagonist activity of a potential SARM is also measured in a normal, nontumor cell line. Examples of normal, nontumor cells lines useful in this method include, but are not limited to, primary rat prostate epithelial and stromal cells, murine muscle cell line C2C12, primary guinea pig smooth muscle cells, primary smooth-muscle cells from immature (I-PSMC) or adult (A-PSMC) rat penis, primary rabbit smooth muscle cell line, prostatic smooth muscle cell line PS-1, prostatic smooth muscle cell line PSMC 1, mouse bone cell cultures and osteoblasts cells and primary rat seminal vesicle lines SVC-1 and SCV-2. Such cell lines are described in the following exemplary references and the references contained therein: Nemeth et. al. J. Andrology 19, 718-724 (1998), Zhang et. al. J. Steroid Biochem. Mol. Biol. 41, 693-696 (1992), Zhang et. al. Prostate 30, 117-129 (1997), Ricciardelli et. al. J. Endocrinol. 140, 373-383 (1994), Gonzalez-Cadavid et. al. Mol. Cell. Endocrinol. 90, 219-229 (1993), Sadeghi-Nejad et. al. Int. J. Impotence Res. 10, 165-169 (1998), Gerdes et. al. Endocrinology 139, 3569-3577 (1998), Sarah et. al. J Cell. Physiol. 185, 416424 (2000), Chen et. al., FEBS Letters 491, 91-93 (2001) and Tajana et. al. EMBO J. 3, 637-644 (1984).

Alternatively, the agonist and antagonist effects of SARMs are measured in nontumor tissues via a series of in vivo rat models in which surrogate endpoints are measured in tissues including, but not limited to, the prostate, seminal vesicle, and levitor ani muscle, as well as the hypothalmic axis via measurement of plasma luteinizing hormone (LH) levels. Several surrogate endpoint in vivo assays can also be utilized to examine the effects of agents on the AR pathway. These assays involve measuring the effects of agents on normal androgen dependent tissues and functions, such as, but not limited to, prostate, seminal vesicle, levator ani muscle, bone, libido, fertility and hypothalamus (measurement of blood LH levels). These assays are widely recognized as having a direct correlation to the effects of the agents on the AR pathways in humans. Some examples of such surrogate endpoint in vivo assays can be found in, but are not limited to, the following references and the references contained therein: Ashby et. al. J. Appl. Tox. 20, 35-47 (2000), Yamada et. al. Tox. Sciences 53, 289-296 (2000), Hamann et. al. J. Med. Chem. 41, 623-639 (1998), Furr et. al. Eur. Urol 29, 83-95 (1996), Broulik et. al. Bone 20, 473-475 (1997), Wang et. al. Poster #P3-126, Endocrine Society 80^(th) Annual Meeting (1998), Hamann et. al. Presentation #S39-2, Endocrine Society 80^(th) Annual Meeting (1998), Maucher et. al. J. Cancer Res. Clin. Oncol. 119, 669-674 (1993), and Risek et al. Presentation #P1-497, Endocrine Society 83^(rd) Annual Meeting (2001).

Animal models bearing a hormone-dependent tumor can also be used to assess the antagonist activity of a potential SARM against the tumor and the agonist or antagonist activity against AR containing normal nontumor tissues in the animal. For example, the above surrogate endpoint in vivo assays can be run using a rat bearing an androgen-dependent rat prostate tumor, such as the Dunning R-3327. In this manner, effects of a SARM on a rat androgen-dependent prostate tumor can be determined while simultaneously examining the effects of the SARM agent on AR containing normal nontumor tissues such as, but not limited to, prostate, seminal vesicle, and levitor ani muscle as well as effects on the hypothalmic axis via measurements of plasma LH levels. In a similar fashion, immune compromised nude rats bearing human androgen-dependent prostate tumors can be employed. In this manner, effects of a SARM on a human androgen-dependent prostate tumor can be determined while simultaneously examining the effects of the SARM agent on normal tissues such as, but not limited to, prostate, seminal vesicle, and levitor ani muscle as well as effects on the hypothalmic axis via measurements of plasma LH levels. In addition, in vivo rat assays can be employed to determine the effect of SARMs on libido and reproduction.

SARMs having antagonist activity in hormone-induced tumors and no activity, or more preferably agonist activity, in other nontumor tissues can also be designed using information about the AR crystal structure and the estrogen receptor (ER) crystal structure with estradiol, tamoxifen or raloxifen. The crystal structure of the androgen receptor ligand binding domain (AR-LBD) has been determined to 2.0 Å resolution and is described in U.S. patent application Ser. No. 09/687,609, filed Oct. 13, 2000 and corresponding PCT/US00/28495, Matias et al., J. Biol. Chem. 275, 26164-26171 (2000) and Sack et. al., Proc. Natl. Acad. Sci. USA 98, 4904-4909 (2001), herein incorporated by reference. The crystal structure of ER is disclosed, for example, in WO 99/50658, herein incorporated by reference. Using these crystal structures, structure-based or rational drug design techniques can be used to design, select, and synthesize chemical entities, including the inhibitory and stimulatory SARMs of the present invention.

One particularly useful drug design technique enabled by this invention is iterative drug design. Iterative drug design is a method for optimizing associations between a protein and a compound by determining and evaluating the three-dimensional structures of successive sets of protein/ligand complexes. Those of skill in the art will understand upon this disclosure that association of natural ligands or substrates with the binding pockets of their corresponding receptors or enzymes is the basis of many biological mechanisms of action. The term “binding pocket” as used herein, refers to a region of a molecule or molecular complex, that, as a result of its shape, favorably associates with another chemical entity or compound, i.e. ligand. Similarly, many drugs exert their biological effects through association with the binding pockets of receptors and enzymes. Such associations may occur with all or any parts of the binding pockets. An understanding of such associations will help lead to the design of drugs having more favorable associations with their target receptor or enzyme, and thus, improved biological effects. Therefore, this information is valuable in designing potential SARMs of this invention.

The term “associating with” refers to a condition of proximity between chemical entities or compounds, or portions thereof, i.e. ligands. The association may be non-covalent, wherein the juxtaposition is energetically favored by hydrogen bonding or van der Waals or electrostatic interactions, or it may be covalent.

In iterative drug design, crystals of a series of protein/ligand complexes are obtained. The three-dimensional structures of each complex are then solved. Such an approach provides insight into the association between the proteins and ligands of each complex. This is accomplished by selecting compounds with inhibitory activity, obtaining crystals of this new protein/ligand complex, solving the three dimensional structure of the complex, and comparing the associations between the new protein/ligand complex and previously solved protein/ligand complexes. By observing how changes in the compound effect the protein/ligand associations, these associations may be optimized.

In some cases, iterative drug design is carried out by forming successive protein/ligand complexes and then crystallizing each new complex. Alternatively, a pre-formed protein crystal is soaked in the presence of an inhibitor, thereby forming a protein/ligand complex and obviating the need to crystallize each individual protein/ligand complex.

As used herein, the term “soaked” refers to a process in which the crystal is transferred to a solution containing the compound of interest.

The present invention also provides for computational methods using three-dimensional models of the androgen and/or estrogen receptors that are based on crystals of AR-LBD/AR-LBD ligand complex and/or the ER-LBD/ER-LBD ligand complex. Generally, the computational method of designing receptor ligands determines which amino acid or amino acids of the receptor ligand binding domain interact with at least one chemical moiety of the ligand. A ligand is then docked into the binding site of the receptor LBD using the three dimensional model of a crystallized protein comprising the AR-LBD or ER-LBD. The orientation of the ligand in the binding site is then optimized (vide infra) and is then used to design at least one chemical modification of a chemical moiety of the ligand that produces a second chemical moiety of the ligand structure that either decreases or increases an interaction between the interacting amino acid(s) from the receptor LDB and the second chemical moiety compared to the interaction between the interacting amino acid and the corresponding chemical moiety on the natural hormones.

The computational methods of the present invention are for designing SARMs using such crystal and three-dimensional structural information to generate synthetic ligands that modulate the conformational changes of the androgen receptor's LBD and/or the estrogen receptor's LBD. These computational methods are particularly useful in designing SARMs to the androgen receptor, wherein the SARM has an extended moiety that prevents any one of a number of ligand-induced molecular events that alter the receptor's influence on the regulation of gene expression, such as preventing the normal coordination of the activation domain observed for a naturally occurring ligand or other ligands that mimic the naturally occurring ligand, such as an agonist. Based upon the structures of the ER-LBD complexed with agonist or antagonist (Shiau, et al. Cell 1998 95:927-937; Pike et al. EMBO J. 1999 18(17): 4608-4618) and the structures of AR-LDB coupled with DHT or other ligands (described in U.S. patent application Ser. No. 09/687,609, filed Oct. 13, 2000 and corresponding PCT/US00/28495, incorporated herein in their entirety and Table A as provided herein) it can be determined how to modify chemical compounds so that they specifically interact with AR-LBD amino acids in the binding site. In particular, the above extended moiety can be directed towards helix-12 of the AR structure in such a fashion as to influence the position of this helix as was seen in the structure of ER complexed with tamoxifen or raloxifen and differing from the position of helix-12 seen in the crystal structure of AR bound with DHT or R 1881, a synthetic analog of DHT that is a more potent agonist (Matias, et al., J. Biol. Chem. 275, 26164-26171 (2000)), and the crystal structure of ER bound with estradiol. Residues on helix-12 that may be affected or in contact with a ligand in the AR-LBD binding site include M894, M895, A896, E897, I898, I899, S900, V901, Q902, V903, P904, K905, I906 and L907. The present invention also relates to the three-dimensional crystal structure as defined by the structure coordinates listed in Table A. The crystal structure of the invention preferably contains at least 25%, more preferably at least 50%, more preferably at least 75%, more preferably at least 90%, more preferably at least 95%, more preferably at least 99%, and most preferably all of the coordinates listed in Table A. More preferably, molecule or molecular complexes are provided comprising all or any part of the ligand binding site defined by structure coordinates of AR-LBD amino acids V685, L700, L701, S702, S703, L704, N705, E706, L707, G708, E709, Q711, A735, I737, Q738, Y739, S740, W741, M742, G743, L744, M745, V746, F747, A748, M749, G750, R752, Y763, F764, A765, L768, F770, M780, M787, I869, L873, H874, F876, T877, F878, M894, M895, A896, E897, I898, I899, S900, V901, Q902, V903, P904, K905, I906 and L907 according to Table A as provided herein, or a mutant or homologue of said molecule or molecular complex. Most preferred are molecules or molecular complexes comprising all or any part of the ligand binding site defined by structure coordinates of AR-LBD amino acids N705, W741, Q711, R752, F764, T877, M895 and I898, according to Table A, or a mutant or homologue of said molecule or molecular complex.

The term “complex” or “molecular complex” as used herein means AR-LBD or a mutant or homologue of AR-LBD in a covalent or non-covalent association with a chemical entity or ligand.

For purposes of the present invention, by “at least a portion of” it is meant all or any part of the ligand binding site defined by these structure coordinates.

By “mutant or homologue” as used herein it is meant a molecule or molecular complex having a similar structure and/or sequences to AR-LBD. By “similar structure” it is meant a mutant or homologue having a binding pocket that has a root mean square deviation from the backbone atoms of said AR-LBD amino acids of not more than 1.5 Angstroms. By “similar sequence” it is meant a mutant or homologue having 30%, or more preferably 75%, identity with AR-LBD.

The term “root mean square deviation” means the square root of the arithmetic mean of the squares of the deviations from the mean. It is a way to express the deviation or variation from a trend or object. For purposes of this invention, the “root mean square deviation” defines the variation in the backbone of a protein or protein complex from the relevant portion of the backbone of the AR portion of the complex as defined by the structure coordinates described herein.

Once the structure coordinates of a protein crystal have been determined they are useful in solving the structures of other crystals.

Thus, in accordance with the present invention, the structure coordinates of an androgen receptor/ligand complex, and portions thereof is stored in a machine-readable storage medium. Such data may be used for a variety of purposes, such as drug discovery and x-ray crystallographic analysis or protein crystal.

Accordingly, in one embodiment of this invention is provided a machine-readable data storage medium comprising a data storage material encoded with the structure coordinates set forth in Table A. One embodiment utilizes System 10 as disclosed in WO 98/11134, the disclosure of which is incorporated herein by reference in its entirety.

The structure coordinates set forth in Table A can also be used to aid in obtaining structural information about another crystallized molecule or molecular complex. This may be achieved by any of a number of well-known techniques, including molecular replacement.

The structure coordinates set forth in Table A can also be used for determining at least a portion of the three-dimensional structure of molecules or molecular complexes which contain at least some structurally similar features to AR. In particular, structural information about another crystallized molecule or molecular complex may be obtained. This may be achieved by any of a number of well-known techniques, including molecular replacement.

Therefore, in another embodiment this invention provides a method of utilizing molecular replacement to obtain structural information about a crystallized molecule or molecular complex whose structure is unknown comprising the steps of:

-   a) generating an X-ray diffraction pattern from said crystallized     molecule or molecular complex; -   b) applying at least a portion of the structure coordinates set     forth in Table A to the X-ray diffraction pattern to generate a     three-dimensional electron density map of the molecule or molecular     complex whose structure is unknown; and -   c) using all or a portion of the structure coordinates set forth in     Table A to generate homology models of AR-LBD or any other nuclear     hormone receptor ligand binding domain.

Preferably, the crystallized molecule or molecular complex is obtained by soaking a crystal of this invention in a solution.

By using molecular replacement, all or part of the structure coordinates of the AR-LBD/AR-LBD ligand complex provided by this invention or molecular complex whose structure is unknown more quickly and efficiently than attempting to determine such information ab initio.

Molecular replacement provides an accurate estimation of the phases for an unknown structure. Phases are a factor in equations used to solve crystal structures that can not be determined directly. Obtaining accurate values for the phases, by methods other than molecular replacement, is a time-consuming process that involves iterative cycles of approximations and refinements and greatly hinders the solution of crystal structures. However, when the crystal structure of a protein containing at least a homologous portion has been solved, the phases from the known structure provide a satisfactory estimate of the phases for the unknown structure.

Thus, this method involves generating a preliminary model of a molecule or molecular complex whose structure coordinates are unknown, by orienting and positioning the relevant portion of the AR-LBD/AR-LBD ligand complex according to Table A within the unit cell of the crystal of the unknown molecule or molecular complex so as best to account for the observed X-ray diffraction pattern of the crystal of the molecule or molecular complex whose structure is unknown. Phases can then be calculated from this model and combined with the observed X-ray diffraction pattern amplitudes to generate an electron density map of the structure whose coordinates are unknown. This, in turn, can be subjected to any well-known model building and structure refinement techniques to provide a final, accurate structure of the unknown crystallized molecule or molecular complex [E. Lattman, “Use of the Rotation and Translation Functions”, in Meth. Enzymol., 115, pp. 55-77 (1985); M. G. Rossmann, ed., “The Molecular Replacement Method”, Int. Sci. Rev. Set., No. 13, Gordon & Breach, New York (1972)].

The structure of any portion of any crystallized molecule or molecular complex, or mutant, homologue or orphan receptor that is sufficiently homologous to any portion of the AR-LBD/AR-LBD ligand complex can be solved by this method. Along with the aforementioned AR, there also exist a number of receptors for which the activating or deactivating ligands may not be characterized. These proteins are classified as nuclear hormone receptors due to strong sequence homology to AR, and are known as orphan receptors.

The structure coordinates are also particularly useful to solve the structure of crystals of AR-LBD/AR-LBD ligand co-complexed with a variety of chemical entities. This approach enables the determination of the optimal sites for interaction between chemical entities, including interaction of candidate AR inhibitors with the complex. For example, high resolution X-ray diffraction data collected from crystals exposed to different types of solvent allows the determination of where each type of solvent molecule resides. Small molecules that bind tightly to these sites can then be designed and synthesized and tested for their AR inhibition activity.

All of the complexes referred to above may be studied using well-known X-ray diffraction techniques and may be refined versus 1.5-3 Å resolution X-ray data to an R value of about 0.20 or less using computer software, such as X-PLOR [Yale University, 1992, distributed by Molecular Simulations, Inc.; see, e.g., Blundell & Johnson, supra; Meth. Enzymol., vol. 114 & 115, H. W. Wyckoff et al., eds., Academic Press (1985)]. This information may thus be used to optimize known AR agonists, partial agonists, antagonists, partial antagonists and SARMS, and more importantly, to design new AR agonists/antagonists.

Accordingly, the present invention is also directed to a binding site in AR-LBD for an AR-LBD ligand in which a portion of AR-LBD ligand is in van der Walls contact or hydrogen bonding contact with at least one of the following residues: V685, L700, L701, S702, S703, L704, N705, E706, L707, G708, E709, Q711, A735, I737, Q738, Y739, S740, W741, M742, G743, L744, M745, V746, F747, A748, M749, G750, R752, Y763, P764, A765, L768, F770, M780, M787, I869, L873, H874, F876, T877, F878, L880, L881, V889, F891, P892, E893, M894, M895, A896, E897, I898, I899, S900, V901, Q902, V903, P904, K905, I906, or L907 of AR-LBD according to Table A. For purposes of this invention, by AR-LBD binding site it is also meant to include mutants or homologues thereof In a preferred embodiment, the mutants or homologues have at least 25% identity, more preferably 50% identity, more preferably 75% identity, and most preferably 95% identity to residues V685, L700, L701, S702, S703, L704, N705, E706, L707, G708, E709, Q711, A735, I737, Q738, Y739, S740, W741, M742, G743, L744, M745, V746, F747, A748, M749, G750, R752, Y763, F764, A765, L768, F770, M780, M787, I869, L873, H874, F876, T877, F878, L880, L881, V889, F891, P892, E893, M894, M895, A896, E897, I898, I899, S900, V901, Q902, V903, P904, K905, I906, or L907 of AR-LBD binding sites according to Table A.

The present invention is also directed to a machine-readable data storage medium, comprising a data storage material encoded with machine readable data, wherein the data is defined by the structure coordinates of an AR-LBD/AR-LBD ligand according to Table A or a homologue of said complex, wherein said homologue comprises backbone atoms that have a root mean square deviation from the backbone atoms of the complex of not more than 3.0 Å. Preferably, the machine-readable data storage medium, according to the invention, is wherein said molecule or molecular complex is defined by the set of structure coordinates for AR-LBD/AR-LBD ligand according to Table A, or a homologue of said molecule or molecular complex, said homologue having a root mean square deviation from the backbone atoms of said amino acids of not more than 2.0 Å. In a preferred embodiment the machine-readable data storage medium comprises a data storage material encoded with a first set of machine readable data comprising a Fourier transform of at least a portion of the structural coordinates for an AR-LBD/AR-LBD ligand according to Table A; which, when combined with a second set of machine readable data comprising an X-ray diffraction pattern of a molecule or molecular complex of unknown structure, using a machine programmed with instructions for using said first set of data and said second set of data, can determine at least a portion of the structure coordinates corresponding to the second set of machine readable data, said first set of data and said second set of data.

The present invention also provides for computational methods using three dimensional models of the androgen receptor that are based on crystals of AR-LBD/AR-LBD ligand complex. Generally, the computational method of designing an androgen receptor ligand determines which amino acid or amino acids of the AR-LBD interact with a chemical moiety (at least one) of the ligand using a three dimensional model of a crystallized protein comprising the AR-LBD with a bound ligand, and selecting a chemical modification (at least one) of the chemical moiety to produce a second chemical moiety with a structure that either decreases or increases an interaction between the interacting amino acid and the second chemical moiety compared to the interaction between the interacting amino acid and the corresponding chemical moiety on the natural hormone. In a preferred embodiments, the method for identifying a compound that modulates androgen receptor activity comprises any combination of the following steps:

-   -   a. modeling test compounds that fit spatially into the AR-LBD as         defined by structure coordinates according to Table A, or using         a three-dimensional structural model of AR-LBD, mutant AR-LBD or         AR-LBD homologue or portion thereof;     -   b. using the AR-LBD structure coordinates or ligand binding site         as set forth herein to identify structural and chemical         features;     -   c. employing identified structural or chemical features to         design or select compounds as potential SARMs;     -   d. employing the three-dimensional structural model or the         ligand binding site to design or select compounds as potential         SARMs;     -   e. synthesizing the potential SARMs;     -   f. screening the potential SARMs in an assay characterized by         binding of a test compound to the AR-LBD; and     -   g. modifying or replacing one or more amino acids from AR-LBD         selected from the group consisting of V685, L700, L701, S702,         S703, L704, N705, E706, L707, G708, E709, Q711, A735, I737,         Q738, Y739, S740, W741, M742, G743, L744, M745, V746, F747,         A748, M749, G750, R752, Y763, F764, A765, L768, F770, M780,         M787, I869, L873, H874, F876, T877, F878, L880, L881, V889,         F891, P892, E893, M894, M895, A896, E897, I898, I899, S900,         V901, Q902, V903, P904, K905, I906 or L907 of

LBD according to Table A.

The computational methods of the present invention are for designing androgen receptor synthetic ligands using such crystal and three dimensional structural information to generate synthetic ligands that modulate the conformational changes of the androgen receptor's LBD. These computational methods are particularly useful in designing an agonist, partial agonist, antagonist or partial antagonist or SARM to the androgen receptor, wherein the agonist, partial agonist, antagonist or partial antagonist or SARM has an extended moiety that prevents any one of a number of ligand-induced molecular events that alter the receptor's influence on the regulation of gene expression, such as preventing the normal coordination of the activation domain observed for a naturally occurring ligand or other ligands that mimic the naturally occurring ligand, such as an agonist. As described herein, synthetic ligands of the androgen receptor will be useful in modulating androgen receptor activity in a variety of medical conditions.

It is also possible to design an extended chemical moiety that is directed towards helix-3 or helix-11 that stabilize or disrupt critical ligand-receptor interactions (Humm et al. Arch. Pharm. (Weinheim) 1990 323:83-87; Poujol, et al. J. Biol. Chem. 2000 275(31):24022-24031). The structure of AR-LBD complexed with DHT shows that the 17α-hydroxyl group of androgens (DHT) forms critical hydrogen bonds with Thr-877 and Asn-705 of the AR-LBD (Sack et al. Proc. Natl Acad. Sci. (USA) 98(9):4904-4909 (2001)). Experiments show that when Asn-705 is mutated to alanine (N705A), that nonsteroidal antiandrogens have low antagonistic properties compared to wild type AR. Asn-705 thus plays a crucial role in the anchoring of nonsteroidal antiandrogens and design of chemical moieties directed at Ans-705 and Thr-877 can also be used for development of a SARM. As described herein, synthetic ligands of the androgen receptor will be useful in inhibiting androgen receptor activity in hormone-induced tumors and activating androgen receptors in other androgen receptor containing nontumor tissues.

The location of the secondary structure (SS) elements for three dimensional structures of ER-LDB and AR-LBD are depicted below. The amino acid sequence alignment shown is based upon a structural superposition of the AR-LBD and the ER-LDB. Sequence numbering is for the AR-LBD. H (or G) indicates that a particular amino acid is in a helix, E (or B) indicates a particular amino acid is in a beta strand. The AR-LDB amino acids extend from Ile-672 through His-917 (SEQ ID NO:1) and the ER-LBD amino acids extend from Ser-305 through Arg-548 (SEQ ID NO:2). Helix numbering is indicated below sequence and structure definitions.                       680                 700                 720                 740                        |         |         |         |         |         |         | AR    ---------IFLNVLEAIEPGVVCAGHDNAQPDSFAALLSSLNELGERQLVHVVKWAKALPGFRNLHVDDQMAVIQYSWMG ER    SLALSLTADQMVSALLDAEPPILYSE------FSEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQVHLLECAWLE SS AR         -HHHHHHHH----------------HHHHHHHHHHHHHHHHHHHHHHHHG---GGG--HHHHHHHHHHHHHHH SS ER -------HHHHHHHHHHH---------------HHHHHHHHHHHHHHHHHHHHHHHHG---GGG--HHHHHHHHHHHHHHH                   |                                  |                         |                 Helix-1                            Helix-3                   Helix-4                      760                 780                 800                 820             |         |         |         |         |         |         |         | AR    LMVFAMGWRSFTNVNSRMLYFAPDLVFNEYRMHKSR-MYSQCVRMRHLSQEFGWLQITPQEFLCMKALLLFSIIPVDGLKN ER    ILMIGLVWRSM--EHPGKLLFAPNLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEFVCLKSIILLNSG------- SS AR HHHHHHHHHHHHHH----EE-B--BEE-HHHHH-------HHHHHHHHHHHHHH----HHHHHHHHHHHGG-EEE------ SS ER HHHHHHHHHG--------EE-B--B-EEGGGGGG---HHHHHHHHHHHHHHHHHH---HHHHHHHHHHHHHH---------        |                            |               |                  |      Helix-5                      Helix-6         Helix-7            Helix-8                               840                 860                 880                      |         |         |         |         |         |         | AR    Q---------KFFDELRMNYIKELDRIIA--CAAAAA-ASCSRRFYQLTKLLDSVQPIARELHQFTFDLLIKSHMVSVDFP ER    -VYTFTLKSLEEKDHIHRVLDKITDTLIHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKGMEHLYSMKCKN---VVPLY SS AR H---------HHHHHHHHHHHHHHHH-------------HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH-HHHH----- SS ER -GGG--HHHHHHHHHHHHHHHHHHHHHHHHHHH----HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH----------                        |                                     |                        Helix-9                                     Helix-10/11             900              |         | AR    EMMAEIISVQVPKILSGKVKPIYFH ER    DLLLEMLD-AHR------------- SS AR HHHHHHHHH-HHHHH---EEE---- SS ER HHHHHHHH-HH--------------           |         Helix-12

Various computer programs are available that use crystallography data such as available for AR and ER and enable the rational design of SARMs of the present invention. Software programs such as ICM (version 2.7 or higher; Molsoft LLC, La Jolla, Calif.) or SYBYL® (Tripos Inc. St Louis, Mo.) can be used with the atomic coordinates from AR and ER crystals to generate three-dimensional models and/or determine the structures involved in ligand binding. Other molecular visualization programs such as INSIGHT II® (Pharmacopeia/Molecular Simulations, Inc., San Diego, Calif.) and GRASP (Columbia University, New York, N.Y.) allow for further manipulation and the ability to introduce new structures. In addition, a number computer modeling systems are available in which the sequence of the AR-LBD and the AR-LBD structure can be entered. The computer system then generates the structural details of the site in which a potential AR modulator binds so that complementary structural details of the potential modulators can be determined. Design in these modeling systems is generally based upon the compound being capable of physically and structurally associating with AR-LBD. In addition, the compound must be able to assume a conformation that allows it to associate with AR-LBD. Some modeling systems estimate the potential inhibitory or binding effect of a potential AR modulator prior to actual synthesis and testing.

Methods for screening chemical entities or fragments for their ability to associate with AR and ER are also well known. Often these methods begin by visual inspection of the active site on the computer screen. Selected fragments or chemical entities are then positioned with the AR-LBD or ER-LBD. Docking is accomplished using software, followed by optimization of the ligand in the receptor binding site by global optimization procedures or molecular dynamics and minimization protocols with standard molecular mechanic forcefields such as CHARMM and AMBER. Examples of computer programs which assist in molecular docking and the selection of chemical fragments or chemical entities useful in the present invention include, but are not limited to, GRID (Goodford, P. J. J. Med. Chem. 1985 28:849-857), AUTODOCK (Goodsell, D. S. and Olsen, A. J. Proteins, Structure, Functions, and Genetics 1990 8:195-202), and DOCK (Kunts et al. J. Mol. Biol. 1982 161:269-288) 10 and ICM (Molsoft LLC, La Jolla Calif., ICM 2.7 Program manual, Abagan et al. 1994 J. Mol. Biol. 235:983-1002, Totrov et al. 1997 Proteins Suppl. 1:215-220).

Upon selection of preferred chemical entities or fragments, their relationship to each other and AR or ER can be visualized and the entities or fragments can be assembled into a single potential modulator. Programs useful in assembling the individual chemical entities include, but are not limited to CAVEAT (Bartlett et al. Molecular Recognition in Chemical and Biological Problems Special Publication, Royal Chem. Soc. 78, 182-196 (1989)) and 3D Database systems (Martin, Y. C. J. Med. Chem. 1992 35:2145-2154).

Alternatively, compounds can be designed de novo using either an empty active site or optionally including some portion of a known inhibitor. Methods of this type of design include, but are not limited to LUDI (Bohm H-J, J. Comp. Aid. Molec. Design 1992 6:61-78) and LeapFrog ™(Tripos Associates, St. Louis. Mo.).

Numerous protocols have been developed to score the designed and docked chemical entities in the receptor binding sites. Programs useful in scoring the chemical entities in protein binding sites include, but are not limited to DOCK (Kunts et al. J. Mol. Biol. 1982 161:269-288, ICM (Molsoft LLC, La Jolla Calif., Totrov et al. 1997 Proteins Suppl. 1:215-220, Schapira et al. 2000 Proc. Natl. Acad. Sci USA 97(3):1008-1013) and SYBYL® (Tripos Inc. St Louis, Mo.

Once a computationally designed ligand (CDL) is synthesized, it can be tested using assays such as those described herein to establish its activity as an antagonist in hormone-dependent tumors and to assess its activity in other nonmalignant AR containing tissues. A CDL, which acts as an antagonist in hormone-dependent tumors and exhibits no activity, or more preferably partial agonist or agonist activity, in other nontumor AR containing tissues is a SARM in accordance with this invention. After such testing, the CDLs can be further refined by generating LBD crystals with a CDL bound to the LBD. The structure of the CDL can then be further refined using established chemical modification methods for three-dimensional models to improve the activity or affinity of the CDL and make second generation CDLs with improved properties, such as that of a “super SARM”, meaning a compound having superior agonist activity while maintaining antagonist activity in selected tissues.

In a particularly preferred embodiment of the present invention, SARMs having the following activity levels are contemplated. Such SARMs are preferred in the methods and compositions of the present invention, and exhibit antagonist activity levels described in the following section (i) and/or (ii), and no activity, or more preferably agonist activity levels described in the following sections (iii) and/or (iv):

(i) an IC₅₀ of about 1 μM or less, more preferably 0.5 μM or less, most preferable 0.1 μM or less relative, to the maximal signal induction obtained for DHT, for the inhibition of at least one hormone-dependent tumor cell line, preferably a prostate tumor cell line or another hormone-dependent tumor cell line predictive of activity in a prostate tumor cell line, such as those described above in connection with screening for SARMs of the present invention and in Examples 3, 4, and 6 below; and/or

(ii) inhibition of hormone dependent tumor growth in vivo in a model such as described above and in Examples 10, 11, 12 or 13 below; and preferably;

(iii) at least 30% activation at 1 μM as compared to DHT, more preferably an EC₅₀ of about 0.5 μM or less, most preferably an EC₅₀ of about 0.1 μM or less, in normal AR-responsive tissue;

(iv) and/or more than 20%, preferably more than 40%, more preferably more than 70%, more preferably more than 90% of an activation effect, in vivo as compared to control animals on the weights of the ventral prostate, seminal vesicles, levator ani and/or luteinizing hormone serum levels as described above and in Example 8. Preferred SARMs of the present invention also act to maintain average normal bone density, average normal muscle mass, average normal reproductive function, and average normal libido seen in ugonadal warm-blooded male mammals, preferably human males. In a preferred embodiment, SARMs of the present invention exhibit antagonist activity against hormone-dependent tumors, while exhibiting no activity or agonist activity against at the same amount or range of amounts. When administered to a patient, this amount or range of amounts is the preferred therapeutically useful range. As will be understood by those of skill in the art upon reading this disclosure, SARMs of the present invention, when used in amounts outside the preferred therapeutically useful range, may exhibit the same antagonist activity against hormone-dependent tumors and no activity or agonist activity against nontumor containing tissues or may no longer exhibit the same specificity or selectivity. For example, SARMs of the present invention, when used in amounts exceeding the preferred therapeutically useful range may exhibit some antagonist activity in nontumor containing tissues.

The present invention is also directed to a selective androgen receptor modulator (SARM), which includes any compound that is an antagonist in hormone-induced tumors and inactive, or more preferably an agonist, in other AR containing nontumor tissues. In a preferred embodiment, the SARM is identified via screening assays as set forth herein or designed in accordance with the computational processes described herein. Compounds, which are small molecules, especially compounds other than peptides or steroids, are preferred. Without limitation to a particular chemotype, compounds selected from the following formulae Ia or Ib are preferred as SARMs of the present invention, especially particular compounds of these formulae set forth in the Examples herein. Compounds of the formula Ia are described further (as compounds of the “formula I”) in U.S. Provisional Patent Application Ser. No. 60/214,392, filed Jun. 28, 2000, U.S. Provisional Patent Application Ser. No. 60/284,617, filed Apr. 18, 2001, and U.S. Patent Application entitled “Fused Cyclic Modulators of Nuclear Hormone Receptor Function”, by Salvati et al., filed Jun. 20, 2001 (Attorney Docket No. LD0191 (NP); compounds of the formula Ib are described further (as compounds of the “formula I”) in U.S. Provisional Pat. Application Ser. No. 60/233,519, filed Sep. 19, 2000, U.S. Provisional Patent Application Ser. No. 60/284,730, filed Apr. 18, 2001, and U.S. Patent Application entitled “Fused Heterocyclic Succinimide Compounds and Analogs Thereof, Modulators of Nuclear Hormone receptor Function”, by Salvati et al., filed Jun. 20, 2001 (Attorney Docket No. LD0192(NP)), all of which applications are incorporated herein by reference in their entirety. Formula Ia is as follows:

where the symbols have the following meanings unless otherwise indicated, and are, for each occurrence, independently selected:

-   -   G is an aryl or heterocyclo (e.g., heteroaryl) group, where said         group is mono- or polycyclic, and which is optionally         substituted at one or more positions, preferably with hydrogen,         alkyl or substituted alkyl, alkenyl or substituted alkenyl,         alkynyl or substituted alkynyl, halo, cycloalkyl or substituted         cycloalkyl, cycloalkenyl or substituted cycloalkenyl, aryl or         substituted aryl, heterocyclo or substituted heterocyclo,         arylalkyl or substituted arylalkyl, heterocycloalkyl or         substituted heterocycloalkyl, CN, R¹OC═O, R¹C═O, R¹C═S, R¹HNC═O,         R¹R²NC═O, HOCR³R^(3′), nitro, R¹OCH₂, R¹O, NH₂, NR⁴R⁵, SR¹,         S═OR¹, SO₂R¹, SO₂OR¹, SO₂NR¹R^(1′), (R¹O)(R^(1′)O)P═O,         (R¹)(R^(1′))P═O, or (R^(1′))(NHR¹)P═O;     -   E is C═Z₂, CR⁷R^(7′) (e.g. CHR⁷), SO₂, P═OR², or P═OOR²;     -   Z₁ is O, S, NH, or NR⁶;     -   Z₂ is O, S, NH, or NR⁶;     -   A₁ is CR⁷ or N;     -   A₂ is CR⁷ or N;     -   Y is J-J′-J″ where J is (CR⁷R^(7′))n and n=0-3, J′ is a bond or         O, S, S═O, SO₂, NH, NR⁶, C═O, OC═O, NR¹C═O, CR⁷R^(7′),         C═CR⁸R^(8′), R²P═O, OPOOR², OPO₂, OSO₂, C═N, NHNH, NHNR⁶, NR⁶NH,         N═N, cycloalkyl or substituted cycoalkyl, cycloalkenyl or         substituted cycloalkenyl, heterocyclo or substituted heterocyclo         or aryl or substituted aryl, and J″ is (CR⁷R^(7 ′))n and n=0-3,         where Y is not a bond;     -   W is CR⁷R^(7′)—CR⁷R^(7′), CR⁸═CR⁸, CR⁷R^(7′)—C═O, NR⁹—CR⁷R^(7′),         N═CR⁸, N═N, NR⁹—NR^(9′), cycloalkyl or substituted cycloalkyl,         cycloalkenyl or substituted cycloalkenyl, heterocyclo or         substituted heterocyclo, or aryl or substituted aryl;     -   Q is H, alkyl or substituted alkyl, alkenyl or substituted         alkenyl, cycloalkyl or substituted cycloalkyl, cycloalkenyl or         substituted cycloalkenyl, heterocycloalkyl or substituted         heterocycloalkyl, arylalkyl or substituted arylalkyl, alkynyl or         substituted alkyl, aryl or substituted aryl, heterocyclo (e.g.,         heteroaryl) or substituted heterocyclo (e.g., substituted         heteroaryl), halo, CN, R¹OC═O, R⁴C═O, R⁵R⁶NC═O, HOCR⁷R^(7′),         nitro, R¹OCH₂, R¹O, NH₂, C═OSR¹, SO₂R¹ or NR⁴R⁵;     -   M is a bond, O, CR⁷R^(7′) or NR¹⁰, and M′ is a bond or NR¹⁰,         with the proviso that at least one of M or M′ must be a bond;     -   L is a bond, (CR⁷R^(7′))n, NH, NR⁵ or N(CR⁷R^(7′))n, where n         =0-3;     -   R¹ and R^(1′) are each independently H, alkyl or substituted         alkyl, cycloalkyl or substituted cycloalkyl, cycloalkenyl or         substituted cycloalkenyl, heterocyclo or substituted         heterocyclo, cycloalkylalkyl or substituted cycloalkyalkyl,         cycloalkenylalkyl or substituted cycloalkenylalkyl,         heterocycloalkyl or substituted heterocycloalkyl, aryl or         substituted aryl, arylalkyl or substituted arylalkyl;     -   R² is alkyl or substituted alkyl, cycloalkyl or substituted         cycloalkyl, cycloalkenyl or substituted cycloalkenyl,         heterocyclo or substituted heterocyclo, cycloalkylalkyl or         substituted cycloalkylalkyl, cycloalkenylalkyl or substituted         cycloalkenylalkyl, heterocycloalkyl or substituted         heterocycloalkyl, aryl or substituted aryl, arylalkyl or         substituted arylalkyl;     -   R³ and R^(3′) are each independently H, alkyl or substituted         alkyl, cycloalkyl or substituted cycloalkyl, cycloalkenyl or         substituted cycloalkenyl, heterocyclo or substituted         heterocyclo, cycloalkylalkyl or substituted cycloalkylalkyl,         cycloalkenylalkyl or substituted cycloalkenylalkyl,         heterocycloalkyl or substituted heterocycloalkyl, aryl or         substituted aryl, arylalkyl or substituted arylalkyl, halo, CN,         hydroxylamine, hydroxamide, alkoxy or substituted alkoxy, amino,         NR¹R², thiol, alkylthio or substituted alkylthio;     -   R⁴ is H, alkyl or substituted alkyl, cycloalkyl or substituted         cycloalkyl, cycloalkenyl or substituted cycloalkenyl,         heterocyclo or substituted heterocyclo, cycloalkylalkyl or         substituted cycloalkylalkyl, cycloalkenylalkyl or substituted         cycloalkenylalkyl, heterocycloalkyl or substituted         heterocycloalkyl, aryl or substituted aryl, arylalkyl or         substituted arylalkyl, R¹C═O, R¹ NHC═O, SO₂OR¹, or SO₂NR¹R^(1′);     -   R⁵ is alkyl or substituted alkyl, cycloalkyl or substituted         cycloalkyl, cycloalkenyl or substituted cycloalkenyl,         heterocyclo or substituted heterocyclo, cycloalkylalkyl or         substituted cycloalkylalkyl, cycloalkenylalkyl or substituted         cycloalkenylalkyl, heterocycloalkyl or substituted         heterocycloalkyl, aryl or substituted aryl, arylalkyl or         substituted arylalkyl, R¹C═O, R¹NHC═O, SO₂R¹, SO₂OR¹, or         SO₂NR¹R^(1′);     -   R⁶ is alkyl or substituted alkyl, cycloalkyl or substituted         cycloalkyl, cycloalkenyl or substituted cycloalkenyl,         heterocyclo or substituted heterocyclo, cycloalkylalkyl or         substituted cycloalkylalkyl, cycloalkenylalkyl or substituted         cycloalkenylalkyl, heterocycloalkyl or substituted         heterocycloalkyl, aryl or substituted aryl, arylalkyl or         substituted arylalkyl, CN, OH, OR¹, R¹C═O, R¹NHC═O, SO₂R¹,         SO₂OR¹, or SO₂NR¹R^(1′);     -   R⁷ and R^(7′) are each independently H, alkyl or substituted         alkyl, alkenyl or substituted alkenyl, cycloalkyl or substituted         cycloalkyl, cycloalkenyl or substituted cycloalkenyl,         heterocyclo or substituted heterocyclo, cycloalkylalkyl or         substituted cycloalkylalkyl, cycloalkenylalkyl or substituted         cycloalkenylalkyl, heterocycloalkyl or substituted         heterocycloalkyl, aryl or substituted aryl, arylalkyl or         substituted arylalkyl, halo, CN, OR¹, nitro, hydroxylamine,         hydroxylamide, amino, NHR⁴, NR²R⁵, NOR¹, thiol, alkylthio or         substituted alkylthio, R¹C═O, R¹OC═O, R¹NHC═O, SO₂R¹, SOR¹,         PO₃R¹R^(1′), R¹R^(1′)NC═O, C═OSR¹, SO₂R¹, SO₂OR¹, or         SO₂NR¹R^(1′);     -   R⁸ and R^(8′) are each independently H, alkyl or substituted         alkyl, alkenyl or substituted alkenyl, cycloalkyl or substituted         cycloalkyl, cycloalkenyl or substituted cycloalkenyl,         heterocyclo or substituted heterocyclo, cycloalkylalkyl or         substituted cycloalkyalkyl, cycloalkenylalkyl or substituted         cycloalkenylalkyl, heterocycloalkyl or substituted         heterocycloalkyl, aryl or substituted aryl, arylalkyl or         substituted arylalkyl, nitro, halo, CN, OR¹, amino, NHR⁴, NR²R⁵,         NOR¹, alkylthio or substituted alkylthio, C═OSR¹, R¹OC═O, R¹C═O,         R¹NHC═O, R¹R^(1′)NC═O, SO₂OR¹, S═OR¹, SO₂R¹, PO₃R¹R^(1′), or         SO₂NR¹R^(1′);     -   R⁹ and R^(9′) are each independently H, alkyl or substituted         alkyl, alkenyl or substituted alkenyl, cycloalkyl or substituted         cycloalkyl, cycloalkenyl or substituted cycloalkenyl,         heterocyclo or substituted heterocyclo, cycloalkylalkyl or         substituted cycloalkylalkyl, cycloalkenylalkyl or substituted         cycloalkenylalkyl, heterocycloalkyl or substituted         heterocycloalkyl, aryl or substituted aryl, arylalkyl or         substituted arylalkyl, CN, OH, OR¹, R¹C═O, R¹OC═O, R¹NHC═O,         SO₂R¹, SO₂OR¹, or SO₂NR¹R^(1′); and     -   R¹⁰ is H, alkyl or substituted alkyl, cycloalkyl or substituted         cycloalkyl, cycloalkenyl or substituted cycloalkenyl,         heterocyclo or substituted heterocyclo, cycloalkylalkyl or         substituted cycloalkylalkyl, cycloalkenylalkyl or substituted         cycloalkenylalkyl, heterocycloalkyl or substituted         heterocycloalkyl, aryl or substituted aryl, arylalkyl or         substituted arylalkyl, CN, OH, OR¹, R¹C═O, R¹OC═O, R¹R^(1′)NC═O,         SO₂R¹, SO₂OR¹, or SO₂NR¹R^(1′).         -   Formula Ib is as follows:             where the symbols have the following meanings unless             otherwise indicated, and are, for each occurrence,             independently selected:     -   G is an aryl or heterocyclo (e.g., heteroaryl) group, where said         group is mono—or polycyclic, and which is optionally substituted         at one or more positions, preferably with hydrogen, alkyl or         substituted alkyl, alkenyl or substituted alkenyl, alkynyl or         substituted alkynyl, halo, cycloalkyl or substituted cycloalkyl,         cycloalkenyl or substituted cycloalkenyl, aryl or substituted         aryl, heterocyclo or substituted heterocyclo, arylalkyl or         substituted arylalkyl, heterocycloalkyl or substituted         heterocycloalkyl, CN, R¹OC═O, R¹C═O, R¹C═S, R¹HNC═O, R¹R²NC═O,         HOCR³R^(3′), nitro, R¹OCH₂, R¹O, NH₂, NR⁴R⁵, SR¹, S═OR¹, SO₂R¹,         SO₂OR¹, SO₂NR¹R^(1′), (R¹O)(R^(1′)O)P═O, oxo, (R¹)(R^(1′))P═O,         or (R^(1′))(NHR¹)P═O;     -   Z₁ is O, S, NH, or NR⁶;     -   Z₂ is O, S, NH, or NR⁶;     -   A₁ is CR⁷ or N;     -   A₂ is CR⁷ or N;     -   Y is J-J′-J″ where J is (CR⁷R^(7′))n and n=0-3, J′ is a bond or         O, S, S═O, SO₂, NH, NR⁷C═O, OC═O, NR¹C═O, CR⁷R^(7′),         C═CR⁸R^(8′), R²P═O, R²P═S, R²OP═O, R²NHP═O, OP═OOR², OP═ONHR²,         OP═OR², OSO₂, C═NR⁷, NHNH, NHNR⁶, NR⁶NH, N═N, cycloalkyl or         substituted cycloalkyl, cycloalkenyl or substituted         cycloalkenyl, heterocyclo or substituted heterocyclo or aryl or         substituted aryl, and J″ is (CR⁷R^(7′))n and n=0-3, where Y is         not a bond;     -   W is CR⁷R^(7′)—CR⁷R^(7′), CR⁸═CR⁸, CR⁷R^(7′)—C═O, NR⁹—CR⁷R^(7′),         N═CR⁸, N═N, NR⁹—NR^(9′), S—CR⁷R^(7′), SO—CR⁷R^(7′),         SO₂—CR⁷R^(7′), cycloalkyl or substituted cycloalkyl,         cycloalkenyl or substituted cycloalkenyl, heterocyclo or         substituted heterocyclo, or aryl or substituted aryl, wherein         when W is not NR⁹—CR⁷R^(7′), N═CR⁸, N═N, NR⁹—NR^(9′),         S—CR⁷R^(7′), SO—CR⁷R^(7′), SO₂—CR⁷R^(7′), or heterocyclo or         substituted heterocyclo, then J′ must be O, S, S═O, SO₂, NH,         NR⁷, OC═O, NR¹C═O, OP═OOR², OP═ONHR², OSO₂, NHNH, NHNR⁶, NR⁶NH,         or N═N;     -   Q₁ is H, alkyl or substituted alkyl, alkenyl or substituted         alkenyl, cycloalkyl or substituted cycloalkyl, cycloalkenyl or         substituted cycloalkenyl, heterocycloalkyl or substituted         heterocycloalkyl, arylalkyl or substituted arylalkyl, alkynyl or         substituted alkynyl, aryl or substituted aryl, heterocyclo         (e.g., heteroaryl) or substituted heterocyclo (e.g., substituted         heteroaryl), halo, CN, R¹OC═O, R⁴C═O, R⁵R⁶NC═O, HOCR⁷R^(7′),         nitro, R¹OCH₂, R¹O, NH₂, C═OSR¹, SO₂R¹ or NR⁴R⁵;     -   Q₂ is H, alkyl or substituted alkyl, alkenyl or substituted         alkenyl, cycloalkyl or substituted cycloalkyl, cycloalkenyl or         substituted cycloalkenyl, heterocycloalkyl or substituted         heterocycloalkyl, arylalkyl or substituted arylalkyl, alkynl or         substituted alkynyl, aryl or substituted aryl, heterocyclo         (e.g., heteroaryl) or substituted heterocyclo (e.g., substituted         heteroaryl), halo, CN, R¹OC═O, R⁴C═O, R⁵R⁶NC═O, HOCR⁷R^(7′),         nitro, R¹OCH₂, R¹O, NH₂, C═OSR¹, SO₂R¹ or NR⁴R⁵;     -   L is a bond, (CR⁷R^(7′))n, NH, NR⁵, NH (CR⁷R^(7′))n, or         NR⁵(CR⁷R^(7′))n, where n =0-3;     -   R¹ and R^(1′) are each independently H, alkyl or substituted         alkyl, cycloalkyl or substituted cycloalkyl, cycloalkenyl or         substituted cycloalkenyl, heterocyclo or substituted         heterocyclo, cycloalkylalkyl or substituted cycloalkyalkyl,         cycloalkenylalkyl or substituted cycloalkenylalkyl,         heterocycloalkyl or substituted heterocycloalkyl, aryl or         substituted aryl, arylalkyl or substituted arylalkyl;     -   R² is alkyl or substituted alkyl, cycloalkyl or substituted         cycloalkyl, cycloalkenyl or substituted cycloalkenyl,         heterocyclo or substituted heterocyclo, cycloalkylalkyl or         substituted cycloalkylalkyl, cycloalkenylalkyl or substituted         cycloalkenylalkyl, heterocycloalkyl or substituted         heterocycloalkyl, aryl or substituted aryl, arylalkyl or         substituted arylalkyl;     -   R³ and R^(3′) are each independently H, alkyl or substituted         alkyl, cycloalkyl or substituted cycloalkyl, cycloalkenyl or         substituted cycloalkenyl, heterocyclo or substituted         heterocyclo, cycloalkylalkyl or substituted cycloalkylalkyl,         cycloalkenylalkyl or substituted cycloalkenylalkyl,         heterocycloalkyl or substituted heterocycloalkyl, aryl or         substituted aryl, arylalkyl or substituted arylalkyl, halo, CN,         hydroxylamine, hydroxamide, alkoxy or substituted alkoxy, amino,         NR¹R², thiol, alkylthio or substituted alkylthio;     -   R⁴ is H, alkyl or substituted alkyl, cycloalkyl or substituted         cycloalkyl, cycloalkenyl or substituted cycloalkenyl,         heterocyclo or substituted heterocyclo, cycloalkylalkyl or         substituted cycloalkylalkyl, cycloalkenylalkyl or substituted         cycloalkenylalkyl, heterocycloalkyl or substituted         heterocycloalkyl, aryl or substituted aryl, arylalkyl or         substituted arylalkyl, R¹C═O, R¹NHC═O, SO₂OR¹, or SO₂NR¹R^(1′);     -   R⁵ is alkyl or substituted alkyl, cycoalkyl or substituted         cycloalkyl, cycloalkenyl or substituted cycloalkenyl,         heterocyclo or substituted heterocyclo, cycloalkylalkyl or         substituted cycloalkylalkyl, cycloalkenylalkyl or substituted         cycloalkenylalkyl, heterocycloalkyl or substituted         heterocycloalkyl, aryl or substituted aryl, arylalkyl or         substituted arylalkyl, R¹C═O, R¹NHC═O, SO₂R¹, SO₂OR¹, or         SO₂NR¹R^(1′);     -   R⁶ is alkyl or substituted alkyl, cycloalkyl or substituted         cycloalkyl, cycloalkenyl or substituted cycloalkenyl,         heterocyclo or substituted heterocyclo, cycloalkylalkyl or         substituted cycloalkylalkyl, cycloalkenylalkyl or substituted         cycloalkenylalkyl, heterocycloalkyl or substituted         heterocycloalkyl, aryl or substituted aryl, arylalkyl or         substituted arylalkyl, CN, OH, OR¹, R¹C═O, R¹NHC═O, SO₂R¹,         SO₂OR¹, or SO₂NR¹R^(1′);     -   R⁷ and R^(7′) are each independently H, alkyl or substituted         alkyl, alkenyl or substituted alkenyl, cycloalkyl or substituted         cycloalkyl, cycloalkenyl or substituted cycloalkenyl,         heterocyclo or substituted heterocyclo, cycloalkylalkyl or         substituted cycloalkylalkyl, cycloalkenylalkyl or substituted         cycloalkenylalkyl, heterocycloalkyl or substituted         heterocycloalkyl, aryl or substituted aryl, arylalkyl or         substituted arylalkyl, halo, CN, OR¹, nitro, hydroxylamine,         hydroxylamide, amino, NHR⁴, NR²R⁵, NOR¹, thiol, alkylthio or         substituted alkylthio, R¹C═O, R¹OC═O, R¹NHC═O, SO₂R¹, SOR¹,         PO₃R¹R^(1′), R¹R^(1′)NC═O, C═OSR¹, SO₂R¹, SO₂OR¹, or         SO₂NR¹R^(1′), or wherein A₁ or A₂ contains a group R⁷ and W         contains a group R⁷, said R7 groups of A₁ or A₂ and W together         form a heterocyclic ring;     -   R⁸ and R^(8′) are each independently H, alkyl or substituted         alkyl, alkenyl or substituted alkenyl, cycloalkyl or substituted         cycloalkyl, cycloalkenyl or substituted cycloalkenyl,         heterocyclo or substituted heterocyclo, cycloalkylalkyl or         substituted cycloalkyalkyl, cycloalkenylalkyl or substituted         cycloalkenylalkyl, heterocycloalkyl or substituted         heterocycloalkyl, aryl or substituted aryl, arylalkyl or         substituted arylalkyl, nitro, halo, CN, OR¹, amino, NHR⁴, NR²R⁵,         NOR¹, alkylthio or substituted alkylthio, C═OSR¹, R¹OC═O, R¹C═O,         R¹NHC═O, R¹R^(1′)NC═O, SO₂OR¹, S═OR¹, SO₂R¹, PO₃R¹R^(1′), or         SO₂NR¹R^(1′); and     -   R⁹ and R^(9′) are each independently H, alkyl or substituted         alkyl, alkenyl or substituted alkenyl, cycloalkyl or substituted         cycloalkyl, cycloalkenyl or substituted cycloalkenyl,         heterocyclo or substituted heterocyclo, cycloalkylalkyl or         substituted cycloalkylalkyl, cycloalkenylalkyl or substituted         cycloalkenylalkyl, heterocycloalkyl or substituted         heterocycloalkyl, aryl or substituted aryl, arylalkyl or         substituted arylalkyl, CN, OH, OR¹, R¹C═O, R¹OC═O, R¹NHC═O,         SO₂R¹, SO₂OR¹, or SO₂NR¹R^(1′).         The present invention is further directed to methods of using         SARMs to inhibit the growth of hormone-induced tumors.         Hormone-induced tumors can be treated by administering to a         patient an effective amount of a SARM with antagonist activity         in hormone-induced tumors and no activity, or more preferably         agonist activity, in other nontumor AR containing tissues. By         “effective amount” it is meant an amount or concentration of         SARM that inhibits the growth of hormone-induced tumor cells in         the patient. In a preferred embodiment, “effective amount” also         refers to an amount or concentration, which induces agonist         activity in nontumor AR containing tissues. Such amounts or         concentrations can be determined routinely by those of ordinary         skill in the art, for example, based upon cell based assays such         as described herein or through other art-recognized means.

SARMs of the present invention can be administered alone or simultaneously or sequentially with radiation and/or one or more active agents, such as chemotherapeutic agents. Examples of classes of anti-cancer and cytotoxic agents useful in combination with the present compounds include but are not limited to: alkylating agents such as nitrogen mustards, alkyl sulfonates, nitrosoureas, ethylenimines, and triazenes; antimetabolites such as folate antagonists, purine analogues, and pyrimidine analogues; antibiotics such as anthracyclines, bleomycins, mitomycin, dactinomycin, and plicamycin; enzymes such as L-asparaginase; farnesyl-protein transferase inhibitors; 5α reductase inhibitors; inhibitors of 17β-hydroxy steroid dehydrogenase type 3; hormonal agents such as glucocorticoids, estrogens/ antiestrogens, androgens/antiandrogens, progestins, and luteinizing hormone-releasing hormone antagonists, octreotide acetate; microtubule-disruptor agents, such as ecteinascidins or their analogs and derivatives; microtubule-stabilizing agents such as taxanes, for example, paclitaxel (Taxol®), docetaxel (Taxotere®), and their analogs, and epothilones, such as epothilones A-F and their analogs; plant-derived products, such as vinca alkaloids, epipodophyllotoxins, taxanes; and topiosomerase inhibitors; prenyl-protein transferase inhibitors; and miscellaneous agents such as hydroxyurea, procarbazine, mitotane, hexamethylmelamine, platinum coordination complexes such as cisplatin and carboplatin; and other agents used as anti-cancer and cytotoxic agents such as biological response modifiers, growth factors; immune modulators and monoclonal antibodies. The compounds of the invention may also be used in conjunction with radiation therapy.

Representative examples of these classes of anti-cancer and cytotoxic agents include but are not limited to mechlorethamine hydrochloride, cyclophosphamide, chlorambucil, melphalan, ifosfamide, busulfan, carmustin, lomustine, semustine, streptozocin, thiotepa, dacarbazine, methotrexate, thioguanine, mercaptopurine, fludarabine, pentastatin, cladribin, cytarabine, fluorouracil, doxorubicin hydrochloride, daunorubicin, idarubicin, bleomycin sulfate, mitomycin C, actinomycin D, safracins, saframycins, quinocarcins, discodermolides, vincristine, vinblastine, vinorelbine tartrate, etoposide, etoposide phosphate, teniposide, paclitaxel, tamoxifen, estramustine, estramustine phosphate sodium, flutamide, buserelin, leuprolide, pteridines, diyneses, levamisole, aflacon, interferon, interleukins, aldesleukin, filgrastim, sargramostim, rituximab, BCG, tretinoin, irinotecan hydrochloride, betamethosone, gemcitabine hydrochloride, altretamine, and topoteca and any analogs or derivatives thereof.

Preferred member of these classes include, but are not limited to, paclitaxel, cisplatin, carboplatin, doxorubicin, carminomycin, daunorubicin, aminopterin, methotrexate, methopterin, mitomycin C, ecteinascidin 743, or porfiromycin, 5-fluorouracil, 6-mercaptopurine, gemcitabine, cytosine arabinoside, podophyllotoxin or podophyllotoxin derivatives such as etoposide, etoposide phosphate or teniposide, melphalan, vinblastine, vincristine, leurosidine, vindesine and leurosine.

Examples of anticancer and other cytotoxic agents include the following: epothilone derivatives as found in German Patent No. 4138042.8; WO 97/19086, WO 98/22461, WO 98/25929, WO 98/38192, WO 99/01124, WO 99/02224, WO 99/02514, WO 99/03848, WO 99/07692, WO 99/27890, WO 99/28324, WO 99/43653, WO 99/54330, WO 99/54318, WO 99/54319, WO 99/65913, WO 99/67252, WO 99/67253 and WO 00/00485; cyclin dependent kinase inhibitors as found in WO 99/24416 (see also U.S. Pat. No. 6,040,321); and prenyl-protein transferase inhibitors as found in WO 97/30992 and WO 98/54966; and agents such as those described generically and specifically in U.S. Pat. No. 6,011,029 (the compounds of which U.S. Patent can be employed together with any NHR modulators (including, but not limited to, those of present invention) such as AR modulators, ER modulators, with LHRH modulators, or with surgical castration, especially in the treatment of cancer).

The combinations of the present invention can also be formulated or co-administered with other therapeutic agents that are selected for their particular usefulness in administering therapies associated with the aforementioned conditions. For example, the compounds of the invention may be formulated with agents to prevent nausea, hypersensitivity and gastric irritation, such as antiemetics, and H₁ and H₂ antihistaminics.

SARMs of the present invention, for example, compounds identified as SARMs by the methods disclosed herein, which are active when given orally can be formulated as liquids, for example, syrups, suspensions or emulsions, tablets, capsules and lozenges. A liquid composition will generally comprise a suspension or solution of the compound in a suitable liquid carrier(s), for example, ethanol, glycerin, sorbitol, non-aqueous solvent such as polyethylene glycol, oils or water, with a suspending agent, preservative, surfactant, wetting agent, flavoring or coloring agent. Alternatively, a liquid formulation can be prepared from a reconstitutable powder. For example, a powder containing active compound, suspending agent, sucrose and a sweetener can be reconstituted with water to form a suspension; and a syrup can be prepared from a powder containing active ingredient, sucrose and a sweetener. A composition in the form of a tablet can be prepared using any suitable pharmaceutical carrier(s), such as those routinely used for preparing solid compositions. Examples of such carriers include magnesium stearate, starch, lactose, sucrose, microcrystalline cellulose, and binders, for example, polyvinylpyrrolidone. The tablet can also be provided with a color film coating, or color included as part of the carrier(s). In addition, active compound can be formulated in a controlled release dosage form as a tablet comprising a hydrophilic or hydrophobic matrix. A composition in the form of a capsule can be prepared, for example, using routine encapsulation procedures, such as by incorporation of active compound and excipients into a hard gelatin capsule. Other examples of capsule preparation include, for example, filling a hard gelatin capsule with a semi-solid matrix of active compound and high molecular weight polyethylene glycol; or filling a soft gelatin capsule with a solution of active compound in polyethylene glycol or a suspension in edible oil, for example liquid paraffin or fractionated coconut oil.

SARMs of the present invention, for example, compounds identified by the methods disclosed herein, which are active when given parenterally, can be formulated, for example, for any suitable mode of parenteral administration, such as for intramuscular or intravenous administration. A typical composition for intra-muscular administration will comprise a suspension or solution of active ingredient in an oil, for example arachis oil or sesame oil. A typical composition for intravenous administration will comprise a sterile isotonic aqueous solution containing, for example, active ingredient, dextrose, sodium chloride, a co-solvent, for example polyethylene glycol and, optionally, a chelating agent, for example ethylenediaminetetracetic acid and an anti-oxidant, for example, sodium metabisulphite. Alternatively, the solution can be freeze dried and then reconstituted with a suitable solvent just prior to administration.

SARMs of the present invention, for example, compounds identified as SARMs by the methods disclosed herein, which are active on rectal administration, can be formulated as suppositories. A typical suppository formulation will generally comprise active ingredient with a binding and/or lubricating agent such as a gelatin or cocoa butter or other low melting vegetable or synthetic wax or fat.

SARMs of the present invention, for example, compounds identified as SARMs by the methods disclosed herein, which are active on topical administration, can be formulated, for example, as transdermal compositions. Such compositions include, for example, a backing, active compound reservoir, a control membrane, liner and contact adhesive.

The typical daily dose of SARM varies according to the activity of the SARM, the individual needs, the condition to be treated and the route of administration. Exemplary suitable doses are in the general range of from 0.001 to 10 mg/kg bodyweight of the recipient per day.

The following nonlimiting examples are provided to further illustrate the present invention.

EXAMPLES Example 1 Exemplary SARMs of the Present Invention

Exemplary SARMS of the present invention are depicted in Table 1. The absolute configuration for the following compounds was not determined. For simplicity in nomenclature, compound [3R-(3aα,4β,7β,7aα)]4-[Octahydro-4-(2-hydroxyethyl)-7-methyl-1 ,3-dioxo-4,7-epoxy-2H-isoindol-2-yl]-1-naphthalenecarbonitrile is designated herein as having an “R” configuration and compound [3aS-(3aα,4β,7β,7aα)]-4-[Octahydro-4-(2-hydroxyethyl)-7-methyl-1,3-dioxo-4,7-epoxy-2H-isoindol-2-yl]-1-naphthalenecarbonitrile is designated herein as having an “S” configuration. Enantiomerically pure products derived from [3aR-(3aα,4β,7β,7aα]-4-[Octahydro-4-(2-hydroxyethyl)-7-methyl-1,3-dioxo-4,7-epoxy-2H-isoindol-2-yl]-1-naphthalenecarbonitrile are designated herein as having an “R” configuration and enantiomerically pure products derived from compound [3aS-(3aα,4β,7β,7aα)]-4[Octahydro-4-(2-hydroxyethyl)-7-methyl-1,3-dioxo-4,7-epoxy-2H-isoindol-2-yl]-1-naphthalenecarbonitrile are designated herein as having an “S” configuration. For simplicity in nomenclature, compound [3aS-(3aα,4β,5β,7β,7aα)]-4-[Octahydro-5-hydroxy-7-(2-hydroxyethyl)-4-methyl-1,3-dioxo-4,7-epoxy-2H-isoindol-2-yl]-1-naphthalenecarbonitrile is designated herein as having an “S” configuration and compound [3aR-(3aα,4β,5β,7β,7aα))-4-[Octahydro-5-hydroxy-7-(2-hydroxyethyl)-4-methyl-1,3-dioxo-4,7-epoxy-2H-isoindol-2-yl]- 1-naphthalenecarbonitrile is designated herein as having an “W” configuration. Enantiomerically pure products derived from [3aS-(3aα,4β,5β,7β,7aα)]-4-[Octahydro-5-hydroxy-7-(2-hydroxyethyl)-4-methyl-1,3-dioxo-4,7-epoxy-2H-isoindol-2-yl]-1-naphthalenecarbonitrile are designated herein as having an “S” configuration and enantiomerically pure products derived from [3aR-(3aα,4β,5β,7β,7aα)]-4-[Octahydro-5-hydroxy-7-(2-hydroxyethyl)4-methyl-1,3-dioxo-4,7-epoxy-2H-isoindol-2-yl]-1-naphthalenecarbonitrile are designated herein as having an “R” configuration. Enantiomerically pure products derived from [3aR-(3aα,4β,5β,7β,7aα)]-4-[7-[2-[[(1,1-Dimethylethyl)dimethylsilyl]oxy]ethyl]octahydro-5-hydroxy-4-methyl-1,3-dioxo-4,7-epoxy-2H-isoindol-2-yl]-2-(trifluoromethyl)-benzonitrile are designated herein as having a “R” configuration and enantiomerically pure products derived from [3aS-(3aα,4β,5β,7β,7aα)]-4-[7-[2-[[(1,1-Dimethyl-ethyl)dimethylsilyl]oxy]ethyl]octahydro-5-hydroxy-4-methyl-1,3-dioxo-4,7-epoxy-2H-isoindol-2-yl]-2-(trifluoromethyl) benzonitrile are designated herein as having an “S” configuration.

The chromatography techniques used to determine the compound retention times of Table 1 are as follows: LCMS=YMC S5 ODS column, 4.6×50 mm eluting with 10-90% MeOH/H20 over 4 minutes containing 0.1% TFA; 4 mL/min, monitoring at 220 nm; LC=YMC S5 ODS column, 4.6×50 mm eluting with 10-90% MeOH/H2O over 4 minutes containing 0.2% phosphoric acid; 4 mL/min, monitoring at 220 nm. The molecular mass of the compounds listed in Table 1, where provided, were determined by MS(ES) by the formula m/z. TABLE 1 Retention Time Cmp Compound Min./Molecular Proc. # Structure Name Mass of Ex. 1

(αR)-α- Methoxybenzeneacetic acid,2- [(3aα,4β,7β,7aα)-2-(4- cyano-1- naphthalenyl)octahydro- 7-methyl-1,3-dioxo- 4,7-epoxy-4H-isoindol- 4-y]ethyl ester. 3.28 & 3.74 LC Atrop Isomers 547.26 [M + Na]⁺ 2f, 2j 2

4-Fluorobenzoic acid, 2-[(3aα,4β,7β,7aα)-2- (4-cyano-1- naphthalenyl)octahydro- 7-methyl-1,3-dioxo- 4,7-epoxy-4H-isoindol- 4-y]ethyl ester. 3.64 & 3.77 LC Atrop Isomers 499.8 [M + Na]⁺ 2f, 2j 3

(3aα,4β,5β,7β,7aα)-7- [2-(4- Fluorophenoxy)ethyl3]hexahydro-5-hydroxy-4- methyl-2-(4-nitro-1- naphthalenyl)-4,7- epoxy-1H-isoindole- 1,3(2H)-dione. 3.53 LC 505.2 [M − H]HU − 2k 4

(3aα,4β,7β,7aα)- Hexahydro-4-[2-(4- methoxyphenoxy)ethyl]- 7-methyl-2-(4-nitro-1- naphthalenyl)-4,7- epoxy-1H-isoindole 1,3(2H)-dione. 3.42 & 3.55 LC Atrop Isomers 503.21 [M + Na]⁺ 5

(3aα,4β,7β,7aα)- Hexahydro-4-methyl-2- (4-nitro-1- naphthalenyl)-7-[2-[4- (trifluoromethyl)pheno xy]ethyl]-4,7-epoxy- 1H-isoindole-1,3(2H)- dione. 3.81 & 3.93 LC Atrop Isomers 563.12 [M + Na]⁺ 2f, 2j 6

(3aα,4β,7β,7aα)- Hexahydro-4-methyl-2- (4-intro-1- naphthalenyl)-7-[2-(4- nitrophenoxy)ethyl]- 4,7-epoxy-1H- isoindole-1,3(2H)- dione. 3.48 & 3.61 LC Atrop Isomers 540.17 [M + Na]⁺ 2f, 2j 7

(3aα,4β,7β,7aα)-4-[2-(4- Fluorophenoxy)ethyl]hexahydro-7-methyl-2- (4-nitro-1- naphthalenyl)-4,7- epoxy-1H-isoindole- 1,3(2H)-dione. 3.48 & 3.61 LC Atrop Isomers 491.46 [M + H]⁺ 2f, 2j 8

(3aα,4β,7β,7aα)-4- [Octahydro-7-methyl-2- (4-nitro-1- dioxo-4,7-epoxy-4H- isoindol-4- yl]ethoxy]benzonitrile. 3.63 LC 498.12 [M + H]⁺ 2f, 2j 9

(3aα,4β,7β,7aα)-4- [Octahydro-4-methyl- 1,3-dioxo-7-[2-[4- (trifluoromethyl)pheno xy]ethyl]-4,7-epoxy- 2H-isoindol-2-yl]-2- (trifluoromethyl)benzo nitrile. 3.93 LC 2f, 2j 10

(3aα,4β,7β,7aα)-4-[2- (Acetyloxy)ethyl]-2-(4- Cyano-1- naphthalenyl)hexahydro- 7-methyl-4,7-epoxy- 1H-isoindole-1,3(2H)- dione. 2.84 & 3.03 LC Atrop Isomers 2f, 2j 11

(3aα,4β,7β,7aα)-4- [Octahydro-4-methyl-7- [2-[(7-methyl-1,2- benzisoxazol-3- yl)oxy]ethyl]-1,3- dioxo-4,7-epoxy-2H- isomdol-2-yl]-1- naphthalenecarbonitrile. 3.79 & 3.92 LC Atrop Isomers 2r 12

(3aα,4β,7β,7aα)-4-[4- [2-(1,2-Benzisoxazol-3- yloxy)ethyl]octahydro- 7-methyl-1,3-dioxo- 4,7-epoxy-2H-isoindol- naphthalenecarbonitrile. 3.55 & 3.70 LC Atrop Isomers 2r 13

(3aα,4β,7β,7aα)-4-[4- [2-[(6-Chloro-1,2- benzisoxazol-3- yl)oxy]ethyl]octahydro- 7-methyl-1,3-dioxo- 4,7-epoxy-2H-isoindol- 2-yl]-1- naphthalenecarbonitrile. 3.89 & 4.02 LC Atrop Isomers 528.0 [M + H]⁺ 2r 14

(3aα,4β,7β,7aα)-4- [Octahydro-4-methyl-7- [2-[(6-nitro-1H- indazol-3- yl)oxy]ethyl]-1,3- dioxo-4,7-epoxy-2H- isomdol-2-yl]-1- naphthalenecarbonitrile. 3.60 & 3.74 LC Atrop Isomers 536.0 [M − H]⁻ 2s 15

(3aα,4β,7β,7aα)-4-[2- (Benzoyloxy)ethyl]-2- (4-cyano-1- naphthalenyl)hexahydro- 7-methyl-4,7-epoxy- 1H-isomdole-1,3(2H)- dione. 3.51 & 3.66 LC Atrop Isomers 2f, 2j 16

(3aα,4β,7β,7aα)-2-(4- Cyano-1-naphthalenyl)- nitrobenzoyl)oxy]ethyl]hexahydro-7-methyl- 4,7-epoxy-1H- isomdole-1,3(2H)- dione. 3.52 & 3.67 LC Atrop Isomers 2f, 2j 17

4-Chlorobenzoic acid, 2-[(3aα,4β,7β,7aα-2- (4-cyano-1- naphthalenyl)octahydro- 7-methyl-1,3-dioxo- 4,7-epoxy-4H-isoindol- 4-y]ethyl ester. 3.79 & 3.83 LC Atrop Isomers 2f, 2j 18

(3aα,4β,7β,7aα)-4-[4- [2-(4- cyanophenoxy)ethyl]- 7-ethyloctalhydro-1,3- dioxo-4,7-epoxy-2H- isoindol-2-yl]-1- naphthalenecarbonitrile. 3.65 LC 492.16 [M + H]+ 2v 19

[3aR-(3aα,4β,7β,7aα)]- Fluorophenoxy)ethyl]octahydro-7-methyl-1,3- dioxo-4,7-epoxy-2H- isoindol-2-yl]-1- naphthalenecarbonitrile. 3.80 LC 471.65 [M + H]⁺ 2n, 2o 20

[3aR-(3aα,4β,7β,7aα)]- 4-[Octahydro-4-]2-(3- methoxyphenoxy)ethyl]- 7-methyl-1,3-dioxo- 4,7-epoxy-2H-isoindol- 2-yl]-1- naphthalenecarbonitrile. 3.73 LC 483.65[M + H]⁺ 2n, 2o 21

[3aR-(3aα,4β,7β,7aα)]- 4-[4-[2-(4- Cyanophenoxy)ethyl]octahydro-7-methyl-1,3- dioxo-4,7-epoxy-2H- isoindol-2-yl]-1- naphthalenecarbonitrile. 3.80 LC 2n, 2o 22

[3aR-(3aα,4β,7β,7aα)]- 4-[Octahydro-4-methyl- 1,3-dioxo-7-[2-]4- (trifluoromethyl)phenoxy]ethyl]-4,7-epoxy- 2H-isomdol-2-yl]-2- (trifluoromethyl)benzo nitrile, faster eluting antipode. 3.93 LC 2i 23

[3aR-(3aα,4β,5β,7β,7aα]- 4-[7-[2-(4- Cyanophenoxy)ethyl]octahydro-5-hydroxy-4- methyl-1,3-dioxo-4,7- epoxy-2H-isomdol-2-yl]-1- naphthalenecarbonitrile. 3.07 LC 494.09 [M + H]⁺ 2p, 2q 24

[3aR-(3aα,4β,5β,7β,7aα]- 4-[7-[2-(4- Chlorophenoxy)ethyl]octahydro-5-hydroxy-4- methyl-1,3-dioxo-4,7- epoxy-2H-isoindol-2-yl]-1- naphthalenecarbonitrile. 3.51 LC 503.08 [M + H]⁺ 2p, 2q 25

[3aR-(3aα,4β,5β,7β,7aα]- 4-[7-[2-(4- Acetylphenoxy)ethyl]octahydro-5-hydroxy-4- methyl-1,3-dioxo-4,7- epoxy-2H-isoindol-2-yl]-1- naphthalenecarbonitrile. 3.05 LC 511.13 [M + H]⁺ 2p, 2q 26

[3aR-(3aα,4β,5β,7β,7aα]- 4-[7-[2-(3- Cyanophenoxy)ethyl]octahydro-5-hydroxy-4- methyl-1,3-dioxo-4,7- epoxy-2H-isomdol-2-yl]-1- naphthalenecarbonitrile. 3.85 LC 494.13 [M + H]⁺ 2p, 2q 27

[3aR-(3aα,4β,5β,7β,7aα]- 4-[Octahydro-5-hydroxy- 4-methyl-1,3-dioxo-7- [2-[(5,6,7,8-tetrahydro-1- naphthalenyl)oxy]ethyl]- 4,7-epoxy-2H-isoindol- 2-yl]-1- naphthalenecarbonitrile. 3.85 LC 523.17 [M + H]⁺ 2p, 2q 28

[3aR-(3aα,4β,5β,7β,7aα]- 4-[Octahydro-5-hydroxy- 4-methyl-1,3-dioxo-7- [2-[(5,6,7,8-tetrahydro- 5-oxo-1- naphthalenyl)oxy]ethyl]- 4,7-epoxy-2H- isoindol-2-yl]-1- naphthalenecarbonitrile. 3.29 LC 537.13 [M + H]⁺ 2p, 2q 29

[3aR-(3aα,4β,5β,7β,7aα]- 4-[7-[2-(1,3-Benzodioxol-5- yloxy)ethyl]octahydro- 5-hydroxy-4-methyl- 1,3-dioxo-4,7-epoxy- 2H-isoindol-2-yl]-1- naphthalenecarbonitrile. 3.22 LC 571.2 [M − H + OAc]⁻ 2p, 2q 30

[3aR-(3aα,4β,5β,7β,7aα]- 4-[7-[2-[(5-Chloro-2- pyridinyl)oxy]ethyl]octahydro-5-hydroxy-4- methyl-1,3-dioxo-4,7- epoxy-2H-isomdol-2-yl]-1- naphthalenecarbontrile. 3.37 LC 504.0 [M + H]⁺ 2p, 2q 31

[3aR-(3aα,4β,5β,7β,7aα]- 4-[7-[2-(1,2-Benzisoxazol- 3-yloxy)ethyl]octahydro- 5-hydroxy-4-methyl- 1,3-dioxo-4,7-epoxy- 2H-isomdol-2-yl)-1- naphthalenecarbontrile 3.29 LC 510.4 [M + H⁺ 2p, 2q 32

[5S-(5α,8α,8aα)]-2-(4- Cyano-1-naphthalenyl)- 4-fluorobenzoyl) tetrahydro-5,8- methanoimidazo[1,5- a]pyrazine-1,3(2H,5H)- dione. 2.76 LC 441.09 [M + H]⁺ 2d 33

[5S-(5α,8α,8aα)]-7-(4- Butylbenzoyl)tetrahydro- 2-(4-nitro-1-naphthalenyl)- 5,8-methanoimidazo]1,5- a]pyrazine-1,3(2H,5H)- dione. 3.69 LC 499.45 [M + H]⁺ 2d 34

[5S-(5α,8α,8aα)]- Hexahydro-2-(4-nitro- 1-naphthalenyl)-1,3- dioxo-5,8- methanoimidazo[1,5- a]pyrazine-7(8H)- carboxylic acid,4- fluorophenyl ester. 3.21 LC 477.38 [M + H]⁺ 2d 35

[3aR-(3aα,4β,7β,7aα)]- 4-[7-[2-[(7-Chloro-4- quinolinyl)oxy]ethyl]octahydro-5-hydroxy-4- methyl-1,3-dioxo-4,7- epoxy-2H-isomdol-2-yl]-1- naphthalenecarbonitrile, trifluoroacetate (1:1). 2.53 LC 554.27 [M + H]⁺ 2p, 2q 36

(3aα,4α,7β,7aα)- Hexahydro-4,7- dimethyl-2-(4-nitro-1- naphthalenyl)-4,7- epoxy-1H-isoindole- 1,3(2H)-dione. 3.04 LCMS 2e 37

[3aR-(3aα,4β,7β,7aα)]-4- [7-[2-(4- Fluorophenoxy)ethyl]octahydro-5-hydroxy4- methyl-1,3-dioxo-4,7- epoxy-2H-isoindol-2-yl]-1- naphthalenecarbomtrile. 3.27 LC 487.11 [M + H]³⁰ 2p, 2q 38

[3aR-(3aα,4β,7β,7aα)]-4- [Octahydro-5-hydroxy- 4-methyl-7-[2-[(4- methyl-2-oxo-2H-1- benzopyran-7- yl)oxy]ethyl]-1,3- dioxo-4,7-epoxy-2H- isomdol-2-yl]-1- naphthalenecarbonitrile 3.15 LC 551.15 [M + H]⁺ 2p, 2q 39

[3aR-(3aα,4β,7β,7aα)]-4- [7-[2-(3,5- Dimethoxyphenoxy)ethyl]octahydro-5-hydroxy- 4-methyl-1,3- dioxo-4,7-epoxy-2H- isoindol-2-yl]-1- naphthalenecarbonitrile. 3.26 LC 529.12 [M + H]⁺ 2p, 2q 40

[3aR-(3aα,4β,7β,7aα)]-4- [7-[2-(4-Chloro-3- methylphenoxy)ethyl]octahydro-5-hydroxy-4- methyl-1,3-dioxo-4,7- epoxy-2H-isoindol-2-yl]-1- naphthalenecarbonitrile 3.68 LC 517.33 [M + H]⁺ 2p, 2q 41

[3aR-(3aα,4β,7β,7aα)]-4- [7-[2-(4-Cyano-2,3- difluorophenoxy)ethyl]octahydro-5-hydroxy- 4-methyl-1,3-dioxo- 4,7-epoxy-2H-isoindol- 2-yl]-1- naphthalenecarbonitrile. 3.23 LC 530.13 [M + H]⁺ 2p, 2q 42

[3aR-(3aα,4β,7β,7aα)]- 4-[7-[2-[(5-Chloro-1,2- benzisoxazol-3- yl)oxy]ethyl]octahydro- 5-hydroxy-4-methyl- 1,3-dioxo-4,7-epoxy- 2H-isomdol-2-yl]-1- naphthalenecarbonitrile. 3.57 LC 602.0 [M − H +OAc]⁻ 2p, 2q 43

[3aR-(3aα,4β,7β,7aα)]- 4-[Octahydro-5- hydroxy-4-methyl-1,3- dioxo-7-[2-[4-(1H- 1,2,4-triazol-1- yl)phenoxy]ethyl]-4,7- epoxy-2H-isoindol-2- yl]-1- naphthalenecarbonitrile. 2.93 LC 536.30 [M + H]⁺ 2p, 2q 44

[3aR-(3aα,4β,7β,7aα)]- 3-[2-[2-(4-Cyano-1- naphthalenyl)octahydro- 6-hydroxy-7-methyl- 1,3-dioxo-4,7-epoxy- 4H-isoindol-4- yl]ethoxy]-5- isoxazolecarboxylic acid, methyl ester. 2.90 LC 518.27 [M + H]⁺ 2p, 2q 46

[3aR-(3aα,4β,5β,7β,7aα)]- 4-[Octahydro-5-hydroxy- 4-methyl-1,3-dioxo-7- [2-[[5- (trifluoromethyl)-2- pyridinyl)oxy]ethyl]- 4,7-epoxy-2H-isoindol- 2-yl]-1- naphthalenecarbonitrile 3.45 LC 538.23 [M + H]⁺ 2p, 2q 47

[3aR-(3α,4β,5β,7β,7aα)]- 4-[Octahydro-5-hydroxy- 4-methyl-7-[2-[4- (1,2,3-thiadiazol-5- yl)phenoxy]ethyl]-1,3- dioxo-4,7-epoxy-2H- isomdol-2-yl]-1- naphthalenecarbonitrile 3.20 LC 553.25 [M + H]⁺ 2p, 2q 48

[3aR-(3aα,4β,5β,7β,7aα)]- 4-[Octahydro-5-hydroxy- 4-methyl-7-[2-[(1- methyl-1H-mdazol-3- yl)oxy]ethyl]-1,3- dioxo-4,7-epoxy-2H- isomdol-2-yl]-1- naphthalenecarbonitrile 3.33 LC 2p, 2q 49

[3aR-(3aα,4β,5β,7β,7aα)]- 4-[7-[2-[(6-Chloro-2- methyl-4- pyrimidinyl)oxy]ethyl]octahydro-5-hydroxy- 4-methyl-1,3-dioxo- 4,7-epoxy-2H-isoindol- 2-yl]-1- naphthalenecarbonitrile 3.02 LC 2p, 2q 50

[3aR-(3aα,4β,5β,7β,7aα)]- 4-[Octahydro-5-hydroxy- 4-methyl-1,3-dioxo-7- [2-[[5- (trifluoromethyl)-2- pyridinyl]oxy]ethyl]- 4,7-epoxy-2H-isomdol- naphthalenecarbomtrile 3.46 LC 558.24 [M + H]⁺ 2p, 2q 60

[3aR-(3aα,4β,5β,7β,7aα)]- N-[4-[2-[2-(4-Cyano-1- naphthalenyl)octahydro- 5-hydroxy-4-methyl- 1,3-dioxo-4,7-epoxy- 7H-isoindol-7- yl]ethoxy]phenyl]acetamide 2.747 LC 526.28 [M + H]⁺ 2p, 2q 61

[3aR-(3aα,4β,5β,7β,7aα)]- 4-[7-[2-(2,4- Dichlorophenoxy)ethyl]octahydro-5-hydroxy- 4-methyl-1,3-dioxo- 4,7-epoxy-2H-isoindol- 2-yl]-1- naphthalenecarbonitrile 3.71 LC 537.17 [M + H]⁺ 2p, 2q 62

[3aR-(3aα,4β,5β,7β,7aα)]- 4-[7-[2-[3,5- Bis(trifluoromethyl) phenoxy]ethyl]octahydro- 5-hydroxy-4-methyl- 1,3-dioxo-4,7-epoxy- 2H-isoindol-2-yl]-1- naphthalenecarbonitrile 3.89 LC 605.25 [M + H]⁺ 2p, 2q 63

[3aR-(3aα,4β,5β,7β,7aα)]- 4-[7-[2-[(5,7-Dichloro-8- quinolinyl)oxy]ethyl]octahydro-5-hydroxy-4- methyl-1,3-dioxo-4,7- epoxy-2H-isoindol-2- yl]-1-naphthalene carbonitrile, trifluoroacetate (1:1) 3.70 LC 588.26 [M + H]⁺ 2p, 2q 64

[3aS-(3aα,4β,5β,7β,7aα)]- 4-[7-[2-[(5-Chloro-1,2- benzisoxazol-3- yl)oxy]ethyl]octahydro- 5-hydroxy-4-methyl- 1,3-dioxo-4,7-epoxy- 2H-isoindol-2-yl]-2- (trifluoromethyl) benzonitrile 3.563 LC 562.08 [M + H]⁺ 2a(i) 65

(3aα,4β,7β,7aβ)-4-[4-[2-(4- Cyanophenoxy)ethyl]- 7-ethyloctahydro-1,3- dioxo-4,7-epoxy-2H- isoindol-2-yl]-1- naphthalenecarbonitrile 3.65 LC 562.08 [M + H]⁺ 2v 66

(3aα,4β,5β,7β,7aα)-4- [7-[2-(4- Cyanophenoxy)ethyl]- 4-ethyloctahydro-5- hydroxy-1,3-dioxo-4,7- epoxy-2H-isoindol-2-yl]-1- naphthalenecarbonitrile 3.15 LC 508.14 [M + H]⁺ 2c(i) 67

[3aR-(3aα,4β,5β,7β,7aα)]- 4-[7-[2-[(5-Chloro-2- pyridinyl)oxy]ethyl]octahydro-5-hydroxy-4- methyl-1,3-dioxo-4,7- epoxy-2H-isoindol-2-yl]-1- (trifluoromethyl) benzonitrile 3.37 LC 522.08 [M + H]⁺ 2a(i)

The in vitro activity of these exemplary SARMs was examined in the MDA MB-453 breast tumor line reporter assay, the Shionogi mouse breast tumor line proliferation assay and C2C 12 muscle cell reporter assay. Both the IC₅₀ (antagonist mode, in the presence of DHT) and the EC₅₀ (agonist mode, in the absence of DHT) relative to the maximal signal obtained by DHT were determined. In addition, for the reporter assays, % activation (absence of DHT) and % inhibition (presence of DHT) at a set drug concentration relative to DHT were determined. For the Shionogi proliferation assay, the % proliferation (absence of DHT) and % inhibition (presence of DHT) of proliferation at a set drug concentration relative to DHT were also determined. Unless indicated in the above Table, compounds were presented as a racemic mixture. While differing in some degree in their level of activity, all the exemplary compounds in the above Table demonstrated a SARM profile in accordance with the present invention.

Specifically, all exemplary SARMs tested exhibited an IC₅₀ of less than 0.8 μM and an EC₅₀ of greater than 5 μM in the MDA MB-453 breast tumor line reporter assay. Similar results were observed in the Shionogi mouse breast tumor line proliferation assay for a number of the SARMs tested. Specifically, several of the compounds exhibited an IC₅₀ of less than 0.8 μM and an EC₅₀ of greater than 5 μM. In contrast, in the C2C 12 muscle cell reporter assay a number of the compounds tested exhibited an IC₅₀ of 3 μM or greater and a particularly preferred subset of the compounds tested exhibited an EC₅₀ of less than 0.8 μM or an agonist activity of greater than 25%. Preferred exemplary SARMs of Table 1, which were tested, include Compounds, 3, 7, 11, 13, 19, 23, 24, 25, 30, 31 and 67.

In addition, the effects of SARMS of Table 1 were compared with full AR antagonists in a Mature Rat Prostate Weight Assay (MRPW, Example 9). To compare the effect of AR full antagonists with SARM Compounds 7, 9, and 36 on ventral prostate (VP), seminal vesicles (SV), levator ani (LA), and Leutinizing hormone (LH) serum levels, mature male rats (n=5) were dosed orally for fourteen consecutive days followed by analysis of organ weights and serum. Compared with the full AR antagonists, in particular, the Compound 2e (prepared in Example 2 below) and Casodex (a known AR antagonist) which showed significant inhibitory effects, SARM Compounds 9 and 36 showed only a modest inhibitory effect on VP, SV, and LA weights at the highest dose (100 mg/kg or “mpk”). Compound 7, also exhibiting only modest inhibition further showed (in contrast with Compounds 9 and 36) a reverse dose response, being even less potent as an inhibitor of VP, SV and LA weights at 100 mpk. The serum LH levels were very similar to the intact controls suggesting very weak, if any, activity of these SARMs at the hypothalamus-pituitary axis.

In vivo agonist activities of the SARM Compounds 7 and 9 of the present invention were also examined in the Rat Levator Ani Muscle Model (Example 8) using a preventative schedule of drug administration. In this animal model, sexually mature (6-8 weeks old) male Sprague-Dawley rats were used. The rats were castrated and broken up into treatment groups and treated with test materials beginning three days following surgery. Potential SARM effects of Compounds 7 and 9 were compared to testosterone via a dose-response study comparing testosterone proprionate (0.3 mg/kg-3 mg/kg) to these SARMs. Both SARMs were tested at 90 mg/kg via oral delivery. Compound 7 increased the levator ani wet weight by 27% compared to the vehicle-treated castrated control while having no effect on the prostate wet weight. Compound 9 was ineffective on both the levator ani muscle and prostate.

Compounds 7 and 9 (which are racemic) were also compared with Casodex in the CWR-22 prostate carcinoma model in nude mice (n=8). All three compounds were administered orally for 14 consecutive days. Both Compounds 7 and 9 exhibited inhibition similar to Casodex (150 mg/kg) when dosed at 75 mpk. The two antipodes of Compound 9 were then separated into Compound 25 (shown in Table 1) and its mirror image Compound 25′ (not shown in Table 1). Both compounds were tested in vivo in the immature wet prostate weight assay. Compound 25 showed little activity in the normal tissues while the full antagonist enantiomer Compound 25′ showed clear activity and a dose response. This is unexpected given the stronger binding affinity and antagonist activity (in MD-453) of Compound 25. Testing of both compounds in the CWR22 human prostate xenograft model showed the opposite activity profile. Compound 25 was as potent as Casodex (150 mg/kg) at a 19 mg/kg dose while Compound 25′ showed no significant activity at the maximum dose tested (75 mpk).

The in vivo data obtained mirrored the in vitro data, showing that the compounds were antagonists to the two AR dependent tumor cell lines while being agonists towards the normal muscle cell line.

Example 2 Chemical Synthesis of SARMs

Exemplary chemical syntheses of SARMs of the present invention are included in the following Examples 2a-2d(i). As will be understood by those of skill in the art upon reading this disclosure, other methods than those set forth herein can also be used in the synthesis of these exemplary SARMs.

The following abbreviations are used herein:

-   -   DBU=1,8-diazabicyclo[5.4.0]undec-7-ene     -   4-DMAP=4-dimethylaminopyridine     -   ee=enantiomeric excess     -   DMF=dimethylformamide     -   EtOAc=ethyl acetate     -   Me=methyl     -   RT=retention time     -   TFA=trifluoroacetic acid     -   THF=tetrahydrofuran     -   TLC=thin layer chromatography     -   pTSA=para-toluenesulfonic acid     -   t-Bu=tert-butyl     -   Ph=phenyl     -   Pd/C=palladium on activated charcoal     -   Ts=tosyl     -   TBS=tert-butyldimethylsilane     -   TEA=triethylamine     -   n-Bu=n-butyl     -   rt=room temperature     -   LC=liquid chromatography     -   Et=ethyl     -   MS=molecular sieves     -   MS(ES)=Electro-Spray Mass Spectrometry     -   DEAD=diethyl azodicarboxylate     -   WSDCC=water soluble dicarbonyldiimide         1-(3-dimethylaminopropyl-3-ethylcarbodiimide hydrochloride     -   TBAF-tetrabutylammonium floride     -   DBAD-Di-tert-butylazodicarboxylate     -   ADDP-1,1-[azodicarbonyl]dipiperdine

Example 2a Production of [5S-(5α,8α,8aα)]-2-[4-Cyano-3-(trifluoromethyl)phenyl]hexahydro-1,3-dioxo-5,8-methanoimidazo[1,5-a]pyrazine-7(8H)-carboxylic acid, 1,1-dimethylethyl ester (2a)

To a solution of 4-isocyanato-2-(trifluoromethyl)-benzonitrile (1.0 mmol) in toluene (4 mL) with activated 4 Å MS (0.300 g) was added (1S-exo)-2,5-diazabicyclo[2.2. 1 ]heptane-2,6-dicarboxylic acid, 2-(1,1-dimethylethyl) 6-methyl ester (2a(1)) (0.220 g, 0.856 mmol) in toluene (6 mL). After 10 h at 25° C., DBU (0.166 mL, 1.11 mmol) was added and the reaction was heated at 81° C. for 2 h. The reaction was then cooled to 25° C. and poured into 1 N HCl (50 mL). The solution was then extracted with methylene chloride (3×30 mL) and the combined organics were dried over anhydrous sodium sulfate. The resulting crude material was purified by flash chromatography on SiO₂ eluting with acetone/chloroform (0-2-4-8% acetone) to give 2a (0.155 g, 42%) MS (ES): m/z 437.09 [M+H]⁺. HPLC RT=3.280 min (100%) (YMC S5 ODS column, 4.6×50 mm; 10-90% MeOH/H₂O gradient, +0.1% TFA; 4 mL/min, 220 nM detection). HPLC RT=3.133 min (100%) (YMC S5 ODS column, 4.6×50 mm; 10-90% MeOH/H₂O gradient,+0.1% TFA; 4 mL/min, 220 nM detection); as white foam.

The starting compound, 2a(1), was made by the following procedure:

N-(tert-butoxycarbonyl)-L-4-hydroxyproline (10.0 g, 43.3 mmol) was dissolved in THF and cooled to 0° C. Borane/THF (1.0 M solution, 86.6 mL) was then added over a 15 min period. The reaction was then warmed to 25° C. followed by heating to reflux for 16 h. The reaction flask was then removed from the heat source and anhydrous methanol (35 mL) was added slowly. After cooling to 25° C., the solvent was removed in vacuo and the resulting crude diol intermediate was taken on directly. The crude diol (1.81 g, 8.34 mmol) was dissolved in methylene chloride (50 mL), 2,6-lutidine (1.46 mL, 12.51 mmol) was added and the mixture was cooled −78° C. tert-Butyl dimethylsilyltrifluoro-methansulfonate (1.92 mL, 8.34 mmol) was then added. After 2h, the mixture was poured into 1 N HCl (100 mL), extracted with methylene chloride (2×100 mL) and the organics were dried over anhydrous sodium sulfate. The resulting crude alcohol was purified by flash chromatography on SiO₂ eluting with acetone in chloroform (0-5-10% acetone) to give 1.011 g (37% for 2-steps) of (2S-trans)-4-hydroxy-2-[[[(1,1-dimethylethyl)dimethylsilyl]oxy]methyl]- 1-pyrrolidinecarboxylic acid, 1,1-dimethylethyl ester (2a(2)) as a clear oil:

2a(2) (3.41 g, 10.3 mmol) was dissolved in anhydrous pyridine (30.0 mL) and cooled to 0° C. p-Toluenesulfonylchloride (5.89 g, 30.9 mmol) was then added in portions over a 10 minute period. The flask was then placed in a refrigerator at 4° C. for 48 h. The resulting solution was poured into 1 N HCl (300 mL), extracted with methylene chloride (3×200 mL) and the organics were dried over anhydrous sodium sulfate. The crude tosylate intermediate was dissolved in THF (50 mL), to which was added H₂O (0.5 mL) followed by pTSA-H₂O (1.03 mmol). Once the reaction was complete as determined by TLC, the mixture was poured into saturated aqueous NaHCO₃ (150 mL) and extracted with methylene chloride (3×50 mL). The combined organics were dried over sodium sulfate. The crude alcohol was purified by flash chromatography on SiO₂ eluting with acetone/chloroform (0-5-10% acetone) to give 2.71 g (71% for 2-steps) of (2S-trans)-2-hydroxymethyl-4-[[(4-methylphenyl)sulfonyl]oxy]-1-pyrrolidinecarboxylic acid, 1,1-dimethylethyl ester (2a(3)) as a clear oil:

To a solution of oxalyl chloride (2.0 M soln in CH₂Cl₂, 2.82 mL) in CH₂Cl₂ (40 mL) at −78° C. was added anhydrous dimethylsulfoxide (0.462 mL, 6.51 mmol). The mixture was allowed to stand for 15 min, after which a solution of 2a(3) (1.61 g, 4.34 mmol) in CH₂Cl₂ (10 mL) was slowly added. After an additional 30 min, triethylamine (1.81 mL, 13.02 mmol) was added and the reaction was slowly warmed to 0° C. The reaction was then quenched with H₂O (25 mL) and diluted with CH₂Cl₂ (100 mL). The mixture was then washed sequentially with 1 N HCl (1×100 mL), saturated aqueous NaHCO₃ (50 mL), and water (2×50 mL). The organics were dried over anhydrous sodium sulfate and the volatile organics removed in vacuo. The crude aldehyde intermediate (1.60 g, 4.34 mmol) was dissolved in THF (25 mL) and diethyl cyanophosphonate (90%, 0.95 mL, 5.64 mmol) was added followed by benzyl amine (1.23 mL, 11.3 mmol). After 2 h, the reaction was complete, as observed by TLC and the volatile organics were removed in vacuo. The crude reaction mixture was purified by flash chromatography on SiO₂ eluting with acetone/chloroform (0-2-3% acetone) to give 1.48 g (70%) of (2S-trans)-2-[cyano[(phenylmethyl)amino]methyl]-4-[[(4-methylphenyl)-sulfonyl]oxy]-1-pyrrolidinecarboxylic acid, 1,1-dimethylethyl ester (2a(4))as a white solid. 2a(4) (structure below) was determined to be a ˜1:1 mixture of diastereomers by NMR spectroscopy.

2a(4) (1.48 g, 3.05 mmol) was dissolved in dichloroethane (25 mL) and diisopropyl ethylamine (1.45 mL) was added. The mixture was heated to 100° C. in a sealed tube for 18 h. The volatiles were then removed in vacuo and the resulting crude material was purified by flash chromatography on SiO₂ eluting with acetone/chloroform (0-2-3% acetone), to yield a mixture of (1S-endo)-6-cyano-5-(phenylmethyl)-2,5-diazabicyclo[2.2. 1 ]heptane-2-carboxylic acid, 1,1-dimethylethyl ester (2a(5A)) (0.591 g, 62%) and (1S-exo)-6-cyano-5-(phenylmethyl)-2,5-diazabicyclo[2.2. 1]heptane-2-carboxylic acid, 1,1-dimethylethyl ester (2a(5B)) (0.370 g, 38%) as clear oils. Structural assignments for these compounds were made after

(2a(5A) (0.400 g, 1.28 mmol) was dissolved in NaOMe (0.5 M, 12.8 mL) and heated to 60° C. for 5 h. The reaction was cooled to 0° C. and 3 N HCl (4.0 mL) was added slowly. After 2h at 0° C. the reaction was poured into saturated aqueous NaHCO₃ (50 mL). The mixture was extracted with CH₂Cl₂ (3×50 mL) and the combined organics were dried over anhydrous sodium sulfate. The crude ester was purified by flash chromatography on SiO₂ eluting with chloroform/acetone (0-2-4% acetone) to give 0.320 g (0.92 mmol, 72%) of (1S-endo)-5-(phenylmethyl)-2,5-diazabicyclo[2.2. 1 ]heptane-2,6-dicarboxylic acid, 2-(1,1-dimethylethyl) 6-methyl ester (2a(6A)) as a clear oil:

2a(5B) (0.400 g, 1.28 mmol) was dissolved in NaOMe (0.5 M, 12.8 mL) and heated to 60° C. for 5 h. The reaction was cooled to 0° C. and 3 N HCl (4.0 mL) was added slowly. After 2h at 0° C. the reaction was poured into saturated aqueous NaHCO₃ (50 mL). The mixture was extracted with CH₂Cl₂ (3×50 mL) and the combined organics were dried over anhydrous sodium sulfate. The crude ester was purified by flash chromatography on SiO₂ eluting with chloroform/acetone (0-2-4% acetone) to give 0.290g (0.85 mmol, 66%) of (1S-exo)-5-phenylmethyl)-2,5-diazabicyclo[2.2. 1 ]heptane-2,6-dicarboxylic acid, 2-(1,1-dimethylethyl) 6-methyl ester (2a(6B)) as a clear oil:

2a(6A) (0.280 g, 0.81 mmol) was dissolved in absolute EtOH (10.0 mL) and Pd/C (10% Pd, 0.080 g) was added. An atmosphere of H₂ was introduced via a balloon and the reaction was stirred at 25° C. for 20 h. The Pd was removed by filtration through celite followed by rinsing with EtOAc. The volatiles were removed in vacuo to give 2a(1) (0.205 g, 99%) as viscous yellow oil. This compound was taken on directly without purification. MS(ES)=m/z 257.18 [M+H]⁺. HPLC RT=1.223 min (95%) (YMC S5 ODS column, 4.6×50 mm; 10-90% MeOH/H₂O gradient,+0.1% TFA; 4 mL/min, 220 nM detection):

Example 2b Production of 5S-(5α,8α,8aα)]-4-(Hexahydro-1,3-dioxo-5,8-methanoimidazo [1,5-a]pyrazin-2(3H)-yl)-2-(trifluoromethyl)benzonitrile (2b)

2a (0.115 g, 0.264 mmol) was dissolved in anhydrous methylene chloride (3 mL) and anhydrous TFA (1.0 mL) was added at 25° C. After 1 h, the reaction was concentrated in vacuo and the resulting residue was dissolved in methylene chloride and poured into saturated aq NaHCO₃. This solution was then extracted with methylene chloride (3×10 mL) and the combined organics dried over anhydrous sodium sulfate. This gave 0.089 g (97%) of free 2b as a yellow solid. MS (ES): m/z 359.09 [M+Na]⁺. HPLC RT=1.477 min (100%) (YMC S5 ODS column, 4.6×50 mm; 10-90% MeOH/H₂O gradient,+0.1% TFA; 4 mL/min, 220 nM detection).

Example 2c Production of [5S-(5α,8α,8aα)]-7-(4-Fluorobenzoyl)tetrahydro-2-(4-nitro-1-naphthalenyl)-5,8-methanoimidazo[1,5-a]pyrazine-1,3(2H,5H)-dione (2c)

[5S-(5α,8α,8aα)]-Tetrahydro-2-(4-nitro-1-naphthalenyl)-5,8 methanoimidazo[1,5-a]pyrazine-1,3(2H,5H)-dione (2c(1)) (0.077 g, 0.228 mmol) was dissolved in methylene chloride (2.0 mL) and TEA (0.127 mL, 0.912 mmol) and 4-DMAP (0.001 g) were added. The reaction was cooled to 0° C. and 4-fluorobenzoylchloride (0.040 mL, 0.342 mmol) was added. The reaction was then slowly warmed to 25° C. After 3 h, the reaction was diluted with methylene chloride (50 mL) and then washed successively with 1N HCl and sat aq NaHCO₃ then and dried over anhydrous sodium sulfate. The crude material was purified by preparative TLC on silica eluting with 5% acetone in chloroform to give 0.022 g of 2c as a yellow solid. HPLC: 100% at 2.960 min (retention time) (YMC S5 ODS column 4.6×50 mm, 10-90% aqueous methanol over 4 minutes containing 0.2% phosphoric acid, 4 mL/min, monitoring at 220 nm), MS (ES): m/z 461.07 [M+H]⁺. The starting material was made as described following.

[5S-(5α,8α,8aα)]-Tetrahydro-2-(4-nitro-1-naphthalenyl)-5,8-methanoimidazo[1,5-a]pyrazine-1,3(2H,5H)-dione (2c(1))

[5S-(5α,8α,8aα)]Hexahydro-2-(4-nitro-1-naphthalenyl)-1,3-dioxo-5,8-methanoimidazo[1,5-a]pyrazine-7(8H)-carboxylic acid, 1,1-dimethylethyl ester (2c(2)) (0.160 g, 0.37 mmol) was dissolved in methylene chloride (5.0 mL) and TFA (1.5 mL) was added at 25° C. After 1.5 h, the reaction was concentrated in vacuo and redissolved in methylene chloride. This solution was washed with sat aq NaHCO₃. The aqueous layer was extracted with methylene chloride (3×25 mL). The combined organics were then dried over anhydrous sodium sulfate. Concentration in vacuo gave 0.115 g of 2c(1) as a yellow solid. HPLC: 93% at 1.747 min (retention time) (YMC S5 ODS column 4.6×50 mm, 10-90% aqueous methanol over 4 minutes containing 0.2% phosphoric acid, 4 mL/min, monitoring at 220 nm). MS (ES): m/z 369.07 [M+MeOH)⁺.

[5S-(5α,8α,8aα)]Hexahydro-2-(4-nitro-1-naphthalenyl)-1,3dioxo-5,8-methanoimidazo[1,5-a]pyrazine-7(8H)-carboxylic acid, 1,1-dimethylethyl ester (2c(2))

2a(1) (0.220 g, 0.856 mmol) was added to a suspension of freshly activated 4 Å molecular sieves (0.300 g) in dry toluene (10.0 mL). To this mixture was added 4-nitronaphthal-1-isocyanate (0.214 g, 1.0 mmol). After stirring at 25° C. for 14 h, DBU (0.166 mL, 1.11 mmol) was added and the reaction was heated at 80° C. for 2 h. After 2h, the reaction was cooled to 25° C. and then poured into 1 N HCl (50 mL). This solution was extracted with methylene chloride (3×30 mL) and the combined organics were dried over anhydrous sodium sulfate. The crude material was purified by flash chromatography on silica eluting with 0-2-6% acetone in chloroform to give 0.211 g of 2c(2) as a yellow foam. HPLC: 95% at 3.130 min (retention time) (YMC S5 ODS column 4.6×50 mm, 10-90% aqueous methanol over 4 minutes containing 0.2% phosphoric acid, 4 mL/min, monitoring at 220 nm). MS (ES): m/z 439.19 [M+H)⁺.

Example 2 Production of [5S-(5α,8α,8aα)]-2-(4-Cyano-1-naphthalenyl)tetrahydro-7-(5-isoxazolylcarbonyl)-5,8-methanoimidazo[1,5-a]pyrazine-1,3(2H,5H)-dione (2dLibSynl), [5S-(5α,8α,8aα)]-2-(4-Cyano-1-naphthalenyl)hexahydro-1,3-dioxo-5,8-methanoimidazo[1,5-a]pyrazine-7(8H)-carboxylic acid, 4-fluorophenyl ester (2dLibSyn2), [5S-(5α,8α,8aα)]-2-(4-Cyano-1-naphthalenyl)tetrahydro-7-[(1-methyl-1H-imidazol-4-yl)sulfonyl]-5,8-methanoimidazo(1,5-a]pyrazine-1,3(2H,5H)-dione (2dLibSyn3) & [5S-(5α,8α,8aα)]-2-(4-Cyano-1-naphthalenyl)-N-(4-fluorophenyl)hexahydro-1,3-dioxo-5,8-methanoimidazo[1,5-a]pyrazine-7(8H)-carboxamide (2dLibSyn4)

Solution Phase Library Synthesis

The below procedure is a general approach to the synthesis of SARMs of the present invention in a solution phase library format. A more detailed description of individual compounds made via this combinatorial approach follows. A series of free amine starting materials analogous to 2c(1) (0.05mmol, prepared as described above) were dissolved in dichloromethane (1.5 mL) in a polystyrene tube with a coarse frit. N,N-(Diisopropyl)aminomethyl polystyrene (3.49 mmol/g, 60 mg) was then added to each reaction vessel followed by addition of the desired acid chloride, isocyanate, chloroformate or sulfonyl chloride (0.10 mmol) in 0.5 mL dichloroethane by automated synthesizer. The reaction vessels were shaken at 25° C. for 24 h and then Tris-(2-Aminoethyl)amine Polystyrene HL (200-400 mesh, 3.3 mmol/g, 75 mg) was added to each reaction vessel and the vessels shaken again for 18 h at 25° C. The liquid from each tube was drained into pretared 2.5 ml STR tubes and the resin was rinsed with dichloromethane (3×0.25 mL). The pretared tubes were then concentrated and analyzed by analytical HPLC and LC-MS. HPLC: (Phenomenex-Prime 5μ C-18 column 4.6×50 mm eluting with 10-90% aqueous methanol over 4 minutes containing 0.1% TFA, 4 mL/min, monitoring at 220 nm).

[5S-(5α,8α,8aα)]-2-(4-Cyano-1-naphthalenyl)tetrahydro-7-(5-isoxazolylcarbonyl)-5,8-methanoimidazo[1,5-a]pyrazine-1,3(2H,5H)-dione (2dLibSynl)

[5S-(5α,8α,8aα)]-4-(Hexahydro-1,3-dioxo-5,8-methanoimidazo[1,5-a]pyrazin-2(3H)-yl)-1-naphthalenecarbonitrile (2d(1)) (0.030 g, 0.094 mmol) was dissolved in dichloromethane (2.0 mL) in a polystyrene tube with a coarse frit. N,N-(Diisopropyl)aminomethyl polystyrene (3.49 mmol/g, 65 mg) was then added to each reaction vessel followed by addition of isoxazolacid chloride (0.025 g, 0.19 mmol) The tube was shaken at 25° C. for 24 h and then Tris-(2-Aminoethyl)amine Polystyrene HL (200-400 mesh, 3.3 mmol/g, 75 mg) was added to the reaction vessel and it was shaken again for 18 h at 25° C. The liquid was drained into pretared 2.5 ml STR tube and the resin was rinsed with dichloromethane (3×0.25 mL). Concentration in vacuo gave the crude 2dLibSyn1 (0.058 g) was a yellow solid. No purification was necessary. HPLC: 100% at 2.237 min (retention time) (YMC S5 ODS column 4.6×50 mm, 10-90% aqueous methanol over 4 minutes containing 0.2% phosphoric acid, 4 mL/min, monitoring at 220 nm). MS (ES): m/z 414.11 [M+H]⁺.

[5S-(5α,8α,8aα)]-2-(4-Cyano-1-naphthalenyl)hexahydro-1,3dioxo-5,8-methanoimidazo[1,5-a]pyrazine-7(8H)-carboxylic acid, 4-fluorophenyl ester (2dLibSyn2)

2d(1) (0.030 g, 0.094 mmol) was dissolved in dichloromethane (2.0 mL) in a polystyrene tube with a coarse frit. N,N-(Diisopropyl)aminomethyl polystyrene (3.49 mmol/g, 65 mg) was then added to each reaction vessel followed by addition of 4-fluorophenylchloroformate (0.033 g, 0. 19 mmol) The tube was shaken at 25° C. for 24 h and then Tris-(2-aminoethyl)amine Polystyrene HL (200-400 mesh, 3.3 mmol/g, 75 mg) was added to the reaction vessel and it was shaken again for 18 h at 25° C. The liquid was drained into a pretared 2.5 ml STR tube and the resin was rinsed with dichloromethane (3×0.25 mL). Concentration in vacuo gave crude 2dLibSyn2 (0.053 g) as a yellow solid. No purification was necessary. HPLC: 93% at 2.987 min (retention time) (YMC S5 ODS column 4.6×50 mm, 10-90% aqueous methanol over 4 minutes containing 0.2% phosphoric acid, 4 mL/min, monitoring at 220 nm). MS (ES): m/z 457.07 [M+H]⁺.

[5S-(5α,8α,8aα)]-2-(4-Cyano-1-naphthalenyI)tetrahydro-7-[(1-methyl-1H-imidazol4-yl)sulfonyl]-5,8-methanoimidazo[1,5-a]pyrazine-1,3(2H,5H)-dione (2dLibSyn3)

25 2d(1) (0.030 g, 0.094 mmol) was dissolved in dichloromethane (2.0 mL) in a polystyrene tube with a coarse frit. N,N-(Diisopropyl)aminomethyl polystyrene (3.49 mmol/g, 65 mg) was then added to each reaction vessel followed by addition of imidazolesulfonylchloride (0.034 g, 0. 19 mmol). The tube was shaken at 25° C. for 24 h and then Tris-(2-aminoethyl)amine Polystyrene HL (200-400 mesh, 3.3 mmol/g, 75 mg) was added to the reaction vessel and it was shaken again for 18 h at 25° C. The liquid was drained into a pretared 2.5 ml STR tube and the resin was rinsed with dichloromethane (3×0.25 mL). Concentration in vacuo gave the crude 2dLibSyn3 (0.043 g) as a yellow solid. No purification was necessary. HPLC: 70% at 1.603 min (retention time) (YMC S5 ODS column 4.6×50 mm, 10-90% aqueous methanol over 4 minutes containing 0.2% phosphoric acid, 4 mL/min, monitoring at 220 nm). MS (ES): m/z 463.07 [M+H]⁺.

[5S-(5α,8α,8aα)]-2-(4-Cyano-1-naphthalenyl)-N-(4-fluorophenyl)hexahydro-1,3-dioxo-5,8-methanoimidazo[1,5-a]pyrazine-7(8H)-carboxamide (2dLibSyn4)

2d(1) (0.030 g, 0.094 mmol) was dissolved in dichloromethane (2.0 mL) in a polystyrene tube with a coarse frit. N,N-(Diisopropyl)aminomethyl polystyrene (3.49 mmol/g, 65 mg) was then added to each reaction vessel followed by addition of 4-fluorophenylisocyanate (0.026 g, 0.19 mmol). The tube was shaken at 25° C. for 24 h and then Tris-(2-aminoethyl)amine Polystyrene HL (200-400 mesh, 3.3 mmol/g, 75 mg) was added to the reaction vessel and it was shaken again for 18 h at 25° C. The liquid was drained into a pretared 2.5 ml STR tube and the resin was rinsed with 20 dichloromethane (3×0.25 mL). Concentration in vacuo gave the crude 2dLibSyn4 (0.058 g) as a yellow solid. No purification was necessary. HPLC: 100% at 2.890 min (retention time) (YMC S5 ODS column 4.6×50 mm, 10-90% aqueous methanol over 4 minutes containing 0.2% phosphoric acid, 4 mL/min, monitoring at 220 nm). MS (ES): m/z 456.4 [M+H]⁺.

Example 2e Production of (3aα,4β,7β,7aα)-4-(Octahydro-4,7-dimethyl-1,3-dioxo4,7-epoxy-2H-isoindol-2-yl)-2-(trifluoromethyl)benzonitrile (2e)

(3aα,4β,7β,7aα)-Hexahydro-4,7-epoxyisobenzofuran-1,3-dione (2e(1))

Freshly distilled dimethyl furan (1.60 mL, 15.3 mmol) was dissolved in CH₂Cl₂ (2.0 mL) and maleic anhydride (1.0 g, 10.2 mmol) was added. The reaction was stirred at 25° C. for 16 h and was then concentrated in vacuo to give a yellow solid. This solid was dissolved in ethyl acetate (30 mL) and Pd/C (10% Pd, 0.200 g) was added. Hydrogen was then introduced by a balloon and the reaction stirred for 24 h. The Pd was removed by filtration through celite rinsing with EtOAc followed by concentration in vacuo to give 2e(1) (1.69 g) as a white solid. 2-Dimensional NOE experiments confirmed the structural assignment to be that of 2e(1).

(3α,4β,7β,7aα)-4-(Octahydro-4,7-dimethyl-1,3-dioxo-4,7-epoxy-2H-isoindol-2-yl)-2-(trifluoromethyl)benzonitrile (2e)

A solution of 2e(1) (640 mg, 3.44 mmol, 1.07 eq) and TsOH (10 mg, cat amount) in toluene (5 mL) was heated in a sealed tube for 2 days. The reaction mixture was cooled to room temperature and then concentrated under reduced pressure. Purification by flash chromatography on silica gel eluting with 50% EtOAc/hexanes gave 400 mg (1.10 mmol, 34%) of 2e as a white solid. HPLC: 99% at 3.04 min (retention time) (YMC S5 ODS column 4.6×50 mm, 10-90% aqueous methanol over 4 minutes containing 0.2% phosphoric acid, 4 mL/min, monitoring at 220 nm), MS (ESI): m/z 382.2 [M+NH₄]⁺.

Example 2f Production of (3aα,4β,7β,7aα)-4-[4-[2-(4-Fluorophenoxy)ethyl]octahydro-7-methyl-1,3-dioxo-4,7-epoxy-2H-isoindol-2-yl]-2-(trifluoromethyl)benzonitrile (2f)

DEAD (0.06 mL, 0.380 mmol, 1.5 eq) was added to a solution of triphenylphosphine (100 mg, 0.380 mmol, 1.5 eq) in THF (1.3 mL) at room temperature under an inert atmosphere. After stirring for 10 mins, 4-fluorophenol (43 mg, 0.380 mmol, 1.5 eq) was added in one portion. The reaction mixture was stirred for 5 mins, (3aα,4β,7β,7aα)-4-[octahydro-4-(2-hydroxyethyl)-7-methyl-1,3-dioxo-4,7-epoxy-2H-isoindol-2-yl]-2-(trifluoromethyl)benzonitrile (2f(1)) (100 mg, 0.254 mmol, 1 eq) was added and stirring was continued for 3.5 h. Purification by flash chromatography on silica gel eluting with 50% EtOAc/Hexanes followed by preparative chromatography [HPLC: 11.93 min (retention time) (YMC S5 ODS column 20×100 mm, 0-100% aqueous methanol over 10 minutes containing 0.1% TFA, 20 mL/min, monitoring at 220 nm)]gave 72 mg (58%) of 2f as a solid. HPLC: 99% at 3.74 min (retention time) (YMC S5 ODS column 4.6×50 mm, 10-90% aqueous methanol over 4 minutes containing 0.2% phosphoric acid, 4 mL/min, monitoring at 220 nm), MS (ESI): m/z 487.1 [M−H]⁻.

The starting compound, 2f(1), was made by the following procedure:

A solution of n-BuLi (83 mL, 133.0 mmol, 1.2 eq, 1.6 M in hexanes) was added to a stirred solution of 2-methylfuran (10 mL, 110.8 mmol, 1 eq) in THF (85 mL) at 0° C. under inert atmosphere. The reaction mixture was stirred for 4 h at room temperature then cooled to 0° C. Ethylene oxide (8.3 mL, 166.3 mmol, 1.5 eq) was added dropwise and the reaction mixture was allowed to warm to room temperature overnight. After quenching with saturated aqueous NH₄Cl, the resulting layers were separated and the aqueous layer was extracted with Et₂O (2×). The combined organic layers were dried over Na₂SO₄ and concentrated under reduced pressure. Distillation at atmospheric pressure (170-185° C.) gave 10.13 g (80.3 mmol, 72%) of 5-methyl-2-finethanol (2f(2)) as a light yellow oil:

A solution of 2f(2) (252 mg, 2 mmol, 1 eq) and 4-(2,5-dihydro-2,5-dioxo-1H-pyrrol-1-yl)-3-trifluoromethylbenzonitrile (798 mg, 3 mmol, 1.5 eq) in CH₂Cl₂ (10 mL) was stirred at room temperature for 2 days. The reaction mixture was concentrated under reduced pressure. Purification by flash chromatography on silica gel eluting with 65% EtOAc/hexanes gave 217 mg of pure (3aα,4β,7β,7aα)-4-[1,3,3a,4,7,7a-hexahydro-4-(2-hydroxyethyl)-7-methyl- 1,3-dioxo-4,7-epoxy-2H-isoindol-2-yl]-2-(trifluoromethyl)benzonitrile (2f(3A)), 73 mg of pure (3aα,4α,7α,7aα)-4-[1,3,3a,4,7,7a-hexahydro-4-(2-hydroxyethyl)-7-methyl-1,3-dioxo-4,7-epoxy-2H-isoindol-2-yl]-2-(trifluoromethyl)benzonitrile (2f(3B)) and 310 mg of a mixture of both 2f(3A) and 2f(3B). All three fractions were isolated as white solids with a total isolated yield of 600 mg (1.53 mmol, 76.5%). 2f(3A): HPLC 90% at 2.56 min (retention time) (YMC S5 ODS column 4.6×50 mm, 10-90% aqueous methanol over 4 minutes containing 0.2% phosphoric acid, 4 mL/min, monitoring at 220 nm). 2f(3B): HPLC 90% at 2.56 min (retention time) (YMC S5 ODS column 4.6×50 mm, 10-90% aqueous methanol over 4 minutes containing 0.2% phosphoric acid, 4 mL/min, monitoring at 220 nm).

A solution of 2f(3A) (0.2 g, 0.51 mmol, 1 eq) and 10% Pd/C (43 mg, cat.) in EtOH (12 mL) was stirred under a hydrogen atmosphere at room temperature for 2 h. The reaction mixture was filtered through celite and concentrated under reduced pressure to give 0.2 g (0.51 mmol, 100%) of 2f(1)as a white solid. HPLC: 95% at 2.59 min (retention time) (YMC S5 ODS column 4.6×50 mm eluting with 10-90% aqueous methanol over 4 minutes containing 0.1% TFA, 4 mL/min, monitoring at 220 nm), MS (ESI): m/z 394.97 [M+H]⁺:

Example 2g Production of (3aα,4β,7β,7aα)-4-[4-(2-Bromoethyl]octahydro-7-methyl-1,3-dioxo-4,7-epoxy-2H-isoindol-2-yl]-2-(trifluoromethyl)benzonitrile (2g)

A solution of 2f(1)(495 mg, 1.26 mmol, 1 eq) and pyridine (0.1 ml, 1.26 mmol, 1 eq) in CH₂Cl₂ (2 ml) was added to a solution of Ph₃PBr₂ (636 mg, 1.51 mmol, 1.2 eq) in CH₂Cl₂ (2 ml) at 0° C. The reaction mixture was stirred at room temperature for 3 hr, then the solvent was removed under reduced pressure. The resulting residue was washed 2× with 10 ml portions of EtOAc-hexane (6:4) and the combined washings were purified by flash chromatography on silica gel eluting with 60% EtOAc/hexane to give 390 mg (0.85 mmol, 67.7%) of 2 g as a white solid. HPLC: 99% at 3.51 min (retention time) (YMC S5 ODS column 4.6×50 mm, 10-90% aqueous methanol over 4 minutes containing 0.2% phosphoric acid, 4 mL/min, monitoring at 220 nm). MS (ESI): m/Z 456.7 [M−H]⁻.

Example 2h Production of (3aα,4β,7β,7aα)-4-[Octahydro-4-(2-hydroxyethyl)-7-methyl-1,3-dioxo-4,7-epoxy-2H-isoindol-2-yl]-2-(trifluoromethyl)benzonitrile (2h)

(3aα,4β,7β,7aα)-4-[4-[2-[[(1,1-Dimethylethyl)dimethylsilyl]oxy]ethyl]octahydro-7-methyl-1,3-dioxo-4,7-epoxy-2H-isoindol-2-yl]-2-(trifluoromethyl)benzonitrile (2h(1)) (0.031 g, 0.061 mmol) was dissolved in THF (0.5 mL) and transferred to a polypropylene container followed by cooling to 0° C. HF•pyridine (˜47% HF, 0.1 mL) was then added. After 15 min, the reaction was complete as determined by LC and was poured into cold sat aqueous NaHCO₃. The mixture was extracted with CH₂Cl₂ (3×10 mL). The combined organic layers were washed with 1 N HCl (1×20 mL) and dried over anhydrous Na₂SO₄. 2 h was isolated as a yellow oil. No purification was necessary

HPLC: 95% at 2.59 min (retention time) (YMC S5 ODS column 4.6×50 mm eluting with 10-90% aqueous methanol over 4 minutes containing 0.1% TFA, 4 mL/min, monitoring at 220 nm). MS (ESI): m/z 394.97 [M+H]⁺.

The starting material, 2h(1), was made by the following procedure.

To a solution of 5-methyl-2-furanethanol 2f(2) (2.00 g, 15.9 mmol) in DMF (50 mL) was added imidazole (1.62 g, 23.9 mmol), followed by tert-butyldimethylsilyl chloride (2.63 g, 17.5 mmol). After 2 h at 25° C., the reaction was poured into diethyl ether (300 mL) and washed with water (1×100 mL), 1N HCl (1×100 mL), water (1×100 mL), brine (1×50 mL) and dried over anhydrous MgSO₄. Crude 2-[2-[[(1,1-dimethylethyl)dimethylsilyl]oxy]ethyl]-5-methylfuran (2h(2)) was analyzed by LCMS and NMR and determined to be pure enough to be carried on directly to the next step. HPLC: 100% at 4.347 min (retention time) (YMC S5 ODS column 4.6×50 mm eluting with 10-90% aqueous methanol over 4 minutes containing 0.1% TFA, 4 mL/min, monitoring at 220 nm):

2h(2) (4.0 g, 18.9 mmol) and maleic anhydride (1.42 g, 14.51 mmol) were dissolved in dichloroethane (10 mL) and stirred at 25° C. for 60 hours. The volatiles were then removed in vacuo and the resulting orange oil was dissolved in absolute ethanol (50 mL) and Pd/C (10% Pd, 1.00 g) was added. Hydrogen was then introduced via a balloon. After 3h, the reaction was filtered through celite rinsing with EtOAc and concentrated in vacuo. The crude anhydride was purified by rapid flash chromatography in SiO₂ eluting with acetone/chloroform (0-2-4% acetone) to give 1.30 g of (3aα,4β,7β,7aα)-4-[2-[[(1,1-dimethylethyl)dimethylsilyl]-oxy]ethyl]hexahydro-7-methyl-4,7-epoxy-1H-isobenzofuran-1,3(2H)-dione (2h(3)) as a clear oil, in addition to 3.00 g of the starting 2-[2-[[(1,1-dimethylethyl)dimethylsilyl]oxy]ethyl]-5-methylfuran. Characterization by proton NMR spectroscopy showed only the exo isomer. 1H NMR, 400 MHz, CDCl3, 3.83 (2 H, t, J=6.0 Hz), 3.22 (1 H, d, J=8.2 Hz), 3.06 (1 H, d, J=8.2 Hz), 1.70-2.25 (6 H, m), 1.55 (3 H, s), 0.82 (9 H, s), 0.00 (6 H, s):

2h(3) (0.250 g, 0.8 mmol) and 4-amino-2-trifluoromethylbenzonitrile (0.124 g, 0.668 mmol) were suspended in dry toluene (2.0 mL) in a sealed tube. MgSO₄ (0.200 g) and triethylamine (0.5 mL) were then added and the tube was sealed and placed in a oil bath at 125° C. After 40 h, the reaction was cooled to 25° C., filtered and concentrated in vacuo. The crude material was purified by flash chromatography on SiO₂ eluting with CH₂Cl₂ to give 0.111 g of 2h(1) as a yellow solid. HPLC: 92% at 4.203 min (retention time) (YMC S5 ODS column 4.6×50 mm eluting with 10-90% aqueous methanol over 4 minutes containing 0.1% TFA, 4 mL/min, monitoring at 220 nm). MS (ESI): m/z 531.1 [M+Na]⁺:

Example 2i Production of [3aR-(3aα,4β,7aα)]-4-[Octahydro-4-methyl-1,3-dioxo-7-[2-[4-(trifluoromethyl)phenoxy]ethyl]4,7-epoxy-2H-isoindol-2-yl]-2-(trifluoromethyl)benzonitrile (2iA), faster eluting antipode and [3aS-(3aα,4β,7β,7aα)]-4-[Octahydro-4-methyl-1,3-dioxo-7-[2-[4-(trifluoromethyl)phenoxy]ethyl]-4,7-epoxy-2H-isoindol-2-yl]-2-(trifluoromethyl)benzonitrile (2iB), slower eluting enantiomer

The racemic compound, synthesized as described for 2f, was separated into the individual antipodes by chiral normal phase liquid chromatography. A Chiralpak AD column (50×500 mm) was used eluting with 85% hexanes/7.5% methanol/7.5% ethanol, @50 mL/min. UV detection at 220 nm was used. The faster eluting isomer isomer 2iA (retention time=55.86 min) was found to have 95.8% ee ([α]_(D) ²⁵=−53.02°, C=3.134 mg/cc in CH₂Cl₂) and the slower eluting isomer 2iB (retention time=62.86 min) was 86% ee ([α]_(D) ²⁵=+48.74°, C=2.242 mg/cc in CH₂Cl₂) by analytical chiral normal phase chromatography.

Example 2j Production of (αR)-α-Methoxybenzeneacetic acid, 2-[(3aα,4β,7β,7aα)-2-(4-cyano-1-naphthalenyl)octahydro-7-methyl-1,3-dioxo-4,7-epoxy-4H-isoindol-4-y]ethyl ester (2j)

(3aα,4β,7β,7aα)-4[-2-[[(1,1-Dimethylethyl)dimethylsilyl]oxy]ethyl]octahydro-7-methyl-1,3-dioxo-4,7-epoxy-2H-isoindol-2-yl]-1-naphthalenecarbonitrile

A solution of 4-amino-1-naphthalenecarbonitrile (19.2 g, 114 mmol) and maleic anhydride (14.0 g, 113 mmol) in AcOH (230 mL) was heated at 115° C. for 12 h. After cooling to rt, the reaction mixture was concentrated under reduced pressure then diluted with CH₂Cl₂ (2.5 L). The organic layer was washed 3× with H₂O(3 L), 1× with sat. aq Na₂CO₃ (1 L) and 1× with brine (1 L), dried over MgSO₄ and concentrated to ˜200 mL under reduced pressure. Purification by flash chromatography on cation exchange resin (60 g, CUBX13M6 from United Chemical Technologies) eluting with CH₂Cl₂ gave 25.0 g (88%) of 4-(2,5-Dihydro-2,5-dioxo-1H-1-yl)-1-naphthalenecarbonitrile as a yellow solid. HPLC 96% at 2.48 min (Phenomenex-prime S5-C18 column 4.6×50 mm, 10-90% aqueous methanol over 4 minutes containing 0.2% phosphoric acid, 4 mL/min, monitoring at 220 nm), MS (ES): m/z 249.25 [M+H]⁺.

4-(2,5-Dihydro-2,5-dioxo-1H-1-yl)-1-naphthalenecarbonitrile (1.00 g, 4.03 mmol) was suspended in benzene (6.0 mL) in a sealed tube and 2h(2) (1.11 g, 5.24 mmol) was added. The reaction was heated at 60° C. for 16 h and then cooled to 25° C. The benzene was removed in vacuo to give a yellow solid. The solid was dissolved in ethyl acetate (40 mL) and Pd/C (10% Pd, 0.300 g) was added. Hydrogen was then introduced via a balloon. After 4 h, the reaction was filtered through celite rinsing with ethyl acetate. Concentration in vacuo gave a pale yellow solid. Which was purified by flash chromatography on silica gel eluting with acetone/chloroform (0% -1.5% -3% acetone) to give 2j(1) (1.53 g) as a yellow foam. HPLC: 86% at 4.173 min (retention time) (YMC S5 ODS column 4.6×50 mm eluting with 10-90% aqueous methanol over 4 minutes containing 0.2% phosphoric acid, 4 mL/min, monitoring at 220 nm).

(3aα,4β,7β,7aα)-4-[Octahydro-4-(2-hydroxyethyl)-7-methyl-1,3-dioxo-4,7-epoxy-2H-isoindol-2-yl]-1-naphthalenecarbonitrile (2j(2))

2j(1) (1.37 g, 2.97 mmol) was dissolved in THF (8.0 mL) and transferred to a polypropylene bottle and cooled to 0° C. HF•Pyridine (2.0 mL) was then added. After 20 min., the reaction was carefully poured into cold sat. aq sodium bicarbonate and extracted with methylene chloride (3×30 mL). The organics were then washed with 1 N HCl and dried over anhydrous sodium sulfate. Concentration in vacuo gave the 2j(2) (0.99 g) as a yellow foam which was not purified further. HPLC: 96% at 2.443 and 2.597 (atropisomers) min (retention time) (YMC S5 ODS column 4.6×50 mm eluting with 10-90% aqueous methanol over 4 minutes containing 0.2% phosphoric acid, 4 mL/min, monitoring at 220 nm), MS (ES): m/z 399.02 [M+Na]⁺.

(αR)-α-Methoxybenzeneacetic acid, 2-[(3aα,4β,7β,7aα)-2-(4-cyano-1-naphthalenyl)octahydro-7-methyl-1,3-dioxo-4,7-epoxy-4H-isoindol-4-y]ethyl ester (2j)

2j(2) (0.200 g, 0.575 mmol) was added to a solution of WSDCC (0.138 g, 0.719 mmol) and (R)-mandelic acid (0.096 g, 0.575 mmol) in dichloromethane (6.0 mL). 4-DMAP (0.005 g) was then added and the reaction stirred at 25° C. for 4 h. The mixture was then diluted with dichloromethane and washed with 1 N HCl (2×10 mL), once with sodium bicarbonate (10 mL) and dried over anhydrous sodium sulfate. Concentration in vacuo gave 2j (0.220 g) as a yellow solid which was not purified further. HPLC: 100% at 3.283 min (retention time) (YMC S5 ODS column 4.6×50 mm eluting with 10-90% aqueous methanol over 4 minutes containing 0.2% phosphoric acid, 4 mL/min, monitoring at 220 nm), MS (ES): m/z 547.26 [M+Na]⁺.

Example 2k Production of (3aα,4β,7β,7aα)-7-[2-[[(1,1-30 Dimethylethyl)dimethylsilyl]oxy]ethyl]hexahydro-5-hydroxy-4-methyl-2-(4-nitro-1-naphthalenyl)4,7-epoxy-1H-isoindole-1,3(2H)-dione (2k)

(3aα,4β,7β,7aα)4-[2-[[(1,1-Dimethylethyl)dimethylsilyl]oxy]ethyl]-3a,4,7,7a-tetrahydro-7-methyl-2-(4-nitro-1-naphthalenyl)-4,7-epoxy-1H-isoindole-1,3(2H)-dione (2k(1))

A solution of 2h(2) (455 mg, 1.894 mmol) and 1-[4-nitronaphthalene]-1H-pyrrole-2,5-dione (254 mg, 0.947 mmol) in benzene (2 mL) was heated at 60° C. overnight. 1-[4-nitronaphthalene]-1H-pyrrole-2,5-dione was made as described in 2j(1). The reaction mixture was concentrated under reduced pressure to give crude 2h(2) as a brown solid, which was used directly in the next step without further purification.

(3aα,4β,5β,7β,7aα)-7-[2-[[(1,1-Dimethylethyl)dimethylsilyl]oxy]ethyl]hexahydro-5-hydroxy-4-methyl-2-(4-nitro-1-naphthalenyl)-4,7-epoxy-1H-isoindole-1,3(2H)-dione (2k)

BH₃•THF (0.95 mL, 0.95 mmol, 1 M solution in THF) was added to a solution of crude 2k(1) (0.95 mmol) in THF (2 mL) at 0° C. After 2k(1) was consumed, the reaction mixture was concentrated under reduced pressure. The resulting residue was then dissolved in toluene (2 mL), Me₃NO (71 mg, 2.84 mmol) was added and the mixture was heated to reflux overnight. The reaction mixture was then cooled to rt, added to H₂O and extracted with EtOAc (3×). The combined organic layers were dried over MgSO₄ and concentrated under reduced pressure. Purification by flash chromatography on SiO₂ eluting with a mixture of 75% EtOAc/30% hexanes, gave 130.2 mg (26%) of 2k as a brown solid. HPLC: 94% at 3.92 min (retention time) (YMC S5 ODS column 4.6×50 mm eluting with 10-90% aqueous methanol over 4 minutes containing 0.2% phosphoric acid, 4 mL/min, monitoring at 220 nm), MS (ES): m/z 527.5 [M+H]⁺.

Example 2l Production of (3aα,4β,5β,7β,7aα)-Hexahydro-5-hydroxy-7-(2-hydroxyethyl)-4-methyl-2-(4-nitro-1-naphthalenyl)-4,7-epoxy-1H-isoindole-1,3(2H)-dione (2l)

A mixture of TBAF (0.3 mL, 0.296 mmol, 1 M solution in THF) and HF (0.3 mL, 50% in H₂O) in CH₃CN (6 mL) was added to a solution of 2k (104 mg, 0.197 mmol) in THF (2 mL) at 0° C. The reaction mixture was stirred overnight at rt. After the starting material was consumed, as was evident by TLC, H₂O and EtOAc were added and the layers were separated. The aqueous layer was extracted with EtOAc (1×) and the combined organic layers were washed with H₂O (1×) and brine (1×), dried over Na₂SO₄ and concentrated under reduced pressure. Purification by flash chromatography on SiO₂ eluting with 5% MeOH/CH₂Cl₂ gave 61.2 mg (75%) of 2l as a yellow solid. HPLC: 99% at 2.47 min (retention time) (YMC S5 ODS column 4.6×50 mm eluting with 10-90% aqueous methanol over 4 minutes containing 0.2% phosphoric acid, 4 mL/min, monitoring at 220 nm), MS (ES): m/z 411.2 [M−H]⁻.

Example 2m Production of (3aα,4β,5β,7β,7aα)-7-[2-(4-Fluorophenoxy)ethyl]hexahydro-5-hydroxy-4-methyl-2-(4-nitro-1-naphthalenyl)-4,7-epoxy-1H-isoindole-1,3(2H)-dione (2m)

DBAD (37.7 mg, 0.164 mmol, 1.5 eq) was added to a solution of PPh₃ (43 mg, 0.164 mmol, 1.5 eq) in THF (1 mL). After stirring for 10 mins, 4-fluorophenol (18.3 mg, 0.164 mmol, 1.5 eq) was added and the reaction mixture was stirred for a further 5 mins. A solution of 2l (45 mg, 0.109 mmol, 1 eq) in THF (1 mL) was added and the mixture was stirred at rt overnight. HPLC showed the crude reaction mixture to contain mostly starting diol (2l), so this mixture was added to a preformed mixture as before of PPh₃ (86 mg, 3 eq), DBAD (75.4 mg, 3 eq) and phenol (36.6 mg, 3 eq) in THF (4 mL) at rt. Stirring was continued until all of 2l was consumed as was evident by HPLC. The reaction was concentrated under reduced pressure. Purification by preparative chromatography [HPLC at 15.2 min (retention time) (YMC S5 ODS A column 20×100 mm, 10-90% aqueous methanol over 15 minutes containing 0.1% TFA, 20 mL/min, monitoring at 220 nm)] gave 25.0 mg (45%) of 2m as a light yellow solid. HPLC: 99% at 3.53 min (retention time) (YMC S5 ODS column 4.6×50 mm eluting with 10-90% aqueous methanol over 4 minutes containing 0.2% phosphoric acid, 4 mL/min, monitoring at 220 nm), MS (ES): m/z 505.2 [M−H]⁻.

DBAD (37.7 mg, 0.164 mmol) was added to a solution of PPh₃ (43 mg, 0.164 mmol) in THF (1 mL). After stirring for 10 mins, 4-fluorophenol (18.3 mg, 0.164 mmol) was added and the reaction mixture was stirred for a further 5 min. A solution of compound 2l (45 mg, 0.109 mmol) in THF (1 mL) was added and the mixture was stirred at rt overnight. HPLC showed the crude reaction mixture to contain mostly starting diol (compound 2l), so this mixture was added to a preformed mixture as before of PPh₃ (86 mg), DBAD (75.4 mg) and phenol (36.6 mg) in THF (4 mL) at rt. Stirring was continued until all of 2l was consumed. The reaction was then concentrated under reduced pressure. Purification by preparative chromatography [HPLC at 15.2 min (retention time) (YMC S5 ODS A column 20×100 mm, 10-90% aqueous methanol over 15 minutes containing 0.1% TFA, 20 mL/min, monitoring at 220 nm)] gave 25.0 mg (45%) of 2m as a light yellow solid. HPLC: 99% at 3.53 min (retention time) (YMC S5 ODS column 4.6×50 mm eluting with 10-90% aqueous methanol over 4 minutes containing 0.2% phosphoric acid, 4 mL/min, monitoring at 220 nm), MS (ES): m/z 505.2 [M−H]⁻.

Example 2n: Production of [3aR-(3aα,4β,7β,7aα)]-4-[Octahydro-4-(2-hydroxyethyl)-7-methyl-1,3-dioxo-4,7-epoxy-2H-isoindol-2-yl]-1-naphthalenecarbonitrile (2nA) & [3aS-(3aα,4β,7β,7aα)]4-[Octahydro-4-(2-hydroxyethyl)-7-methyl-1,3-dioxo-4,7-epoxy-2H-isoindol-2-yl]-1-naphthalenecarbonitrile (2nB)

Racemic 2j(2) was separated into its enantiomers by preparative chiral HPLC (CHIRALPAK AD 5×50 cm column; eluting with 20% MeOH/EtOH (1:1) in heptane (isocratic) at 50 mL/min, (220 nm) to give the faster eluting compound 2nA (Chiral HPLC: 13.54 min; CHIRALPAK AD 4.6×250 mm column; eluting with 20% MeOH/EtOH (1:1) in heptane at 1 mL/min) and the slower eluting compound 2nB (Chiral HPLC: 14.99 min; CHIRALPAK AD 4.6×250 mm column; eluting with 20% MeOH/EtOH (1:1) in heptane at 1 mL/min). The absolute conformation for compounds 2nA and 2nB have not been established. For simplicity in nomenclature, we have designated compound 2nA as having an “R” configuration and compound 2nB as having a “S” configuration. Enantiomerically pure products derived from 2nA will be designated as having a “R” configuration and enantiomerically pure products derived from 2nB will be designated as having a “S” configuration.

Example 2o Production of [3aR-(3aα,4β,7β,7aα)]-4-[4-[2-(3-Fluorophenoxy)ethyl]octahydro-7-methyl-1,3-dioxo-4,7-epoxy-2H-isoindol-2-yl]-1-naphthalenecarbonitrile (2oA) & [3aS-(-(3aα,4β,7β,7aα)]-4-[4-[2-(3-Fluorophenoxy)ethyl]octahydro-7-methyl-1,3-dioxo-4,7-epoxy-2H-isoindol-2-yl]-1-naphthalenecarbonitrile (2oB)

To a solution of triphenylphosphine (0.0524 g, 0.20 mmol) in THF (2.0 mL) was added DBAD (0.046 g, 0.2 mmol). After 10 min, 3-fluorophenol (0.018 mL, 0.2 mmol) was added. After 10 additional minutes, enantiomerically pure 2nA (0.050 g, 0.133 mmol) was added. After 3 h at 25° C., the reaction was concentrated in vacuo and purified by preparative HPLC (YMC S5 ODS 20×100 mm, 10-90% aqueous methanol over 15 minutes containing 0.2% TFA, 20 mL/min, monitoring at 220 nm) to give 0.031 g of compound 2oA as a white solid. This process was repeated with enantiomerically pure compound 2nB to yield 2oB. 2oA: HPLC: 100% at 3.80 min (retention time) (YMC S5 ODS column 4.6×50 mm, 10-90% aqueous methanol over 4 minutes containing 0.2% phosphoric acid, 4 mL/min, monitoring at 220 nm), MS (ES): m/z 471.65 [M+H]⁺, [α]_(D) ²⁵=−47.371 (c=4.412 mg/cc, CH₂Cl₂). 2oB: HPLC: 100% at 3.80 min (retention time) (YMC S5 ODS column 4.6×50 mm, 10-90% aqueous methanol over 4 minutes containing 0.2% phosphoric acid, 4 mL/min, monitoring at 220 nm), MS (ES): m/z 471.65 [M+H]⁺, [α]_(D) ²⁵=+24.3 (c=4.165 mg/cc, CH₂Cl₂).

Example 2p Production of [3aS-(3aα,4β,5β,7β,7aα)]-4-[Octahydro-5-hydroxy-7-(2-hydroxyethyl)-4-methyl-1,3-dioxo-4,7-epoxy-2H-isoindol-2-yl]-1-naphthalenecarbonitrile (2pA) & [3aR-(3aα,4β,5β,7β,7aα)]-4-[Octahydro-5-hydroxy-7-(2-hydroxyethyl)-4-methyl-1,3-dioxo-4,7-epoxy-2H-isoindol-2-yl]-1-naphthalenecarbonitrile (2pB)

(3aα,4β,7β,7aα)-4-[4-[2-[[(1,1-Dimethylethyl)dimethylsilyl]oxy]ethyl]-1,3,3a,4,7,7a-hexahydro-1,3-dioxo-4,7-epoxy-2H-isoindol-2-yl]-1-naphthalenecarbonitrile (2p(1))

4-(2,5-Dihydro-2,5-dioxo-1H-1-yl)-1-naphthalenecarbonitrile (18.3 g, 68.7 mmol) was added to a solution of 2h(2) (26.6 g, 110.6 mmol) in benzene (75 mL) and heated to 60° C. overnight. After cooling to rt, the reaction mixture was concentrated under reduced pressure. The residue was treated with MEOH (250 mL) with stirring at 0° C. for 10 min. The resulting solid was filtered, washed with cold MeOH (2×10 mL) and dried to give 26.7 g (79.5%) of 2p(1) as a yellow solid. HPLC analysis of the above solid revealed it to be 95% pure (HPLC conditions: 95% at 2.48 min (Phenomenex-prime S5-C18 column, 4.6×50 mm, 10%-90% aqueous methanol over 4 minute gradient with 0.2% H₃PO₄, detecting at 220 nm)). The filtrate was then concentrated under reduced pressure and the resulting solid was chromatographed, eluting with 3% acetone/CHCl₃, to give an additional 4.36 g of 2p(1) (13%), giving a total final yield of 92.5%.

(3aα,40β,7β,7aα)-4-[7-[2-[[(1,1-Dimethylethyl)dimethylsilyl]oxy]ethyl]octahydro-5-hydroxy-4-methyl-1,3-dioxo-4,7-epoxy-2H-isoindol-2-yl]-1-naphthalenecarbonitrile (2p(2))

A mixture of 2p(1) (10 g, 20.46 mmol) and RhCl(PPh₃)₃ (0.947 mg, 1.02 mmol) was evacuated and filled with argon (3×). THF (200 mL) was added and once all particulates had dissolved, catecholborane (4.4 mL, 40.93 mmol) was slowly added dropwise. When the formation of product ceased, as was determined by HPLC, the reaction mixture was cooled to 0° C. and quenched with phosphate buffer (330 mL, pH 7.2) then EtOH (130 mL) and H₂O₂ (300 mL, 30% aq sol) were added. Once boronate was consumed, the mixture was extracted with CH₂Cl₂ (3×) and the combined organic layers were washed with 1N NaOH, 10% aq NaHSO₃ (1:1, 1×) and brine (1×). The combined washes was extracted with CH₂Cl₂ (1×) and the combined organic layers were dried over Na₂SO₄. Purification by flash chromatography on silica gel eluting with 10% to 30% acetone/CHCl₃ gradient over 25 min gave 7.1 g (68%) of 2p(2) as a light yellow solid. HPLC conditions: 98% at 3.82 min (Phenomenex-prime S5-C18 column 4.6×50 mm, 10%-90% aqueous methanol over 4 minute gradient with 0.2% H₃PO₄, detecting at 220 nm).

[3aS-(3aα,4β,5β,7β,7aα)]4-[7-[2-[[(1,1-Dimethylethyl)dimethylsilyl]oxy]ethyl]octahydro-5-hydroxy-4-methyl-1,3-dioxo-4,7-epoxy-2H-isoindol-2-yl]-1-naphthalenecarbonitrile (2p(3A)) and [3aR-(3aα,4β,5β,7β,7aα)]4-[7-[2-[[(1,1Dimethylethyl)dimethylsilyl]oxy]ethyl]octahydro-5-hydroxy-4-methyl-1,3-dioxo-4,7-epoxy-2H-isoindol-2-yl]-1-naphthalenecarbonitrile (2p(3B))

The racemic compound 2p(2) was separated into the individual enantiomers by chiral normal phase liquid chromatography. A Chiralpak OD column (50×500 mm) was used, eluting with 13% EtOH/hexanes over 99 min at 50 mL/min detecting at 220 nm. The faster eluting isomer 2p(3A) had a retention time=45 min and the slower eluting isomer 2p(3B) had a retention time=66 min.

[3aS-(3aα,4β,5β,7β,7aα)]-4-[Octahydro-5-hydroxy-7-(2-hydroxyethyl)-4-methyl-1,3-dioxo-4,7-epoxy-2H-isoindol-2-yl]-1-naphthalenecarbonitrile (2pA) and [3aR-(3aα,4β,5β,7β,7aα)]-4-[Octahydro-5-hydroxy-7-(2-hydroxyethyl)-4-methyl-1,3-dioxo-4,7-epoxy-2H-isoindol-2-yl]-1-naphthalenecarbonitrile, (2pB)

2p(3A) (0.84 g, 2.14 mmol) was dissolved in 2% 12 N HCl/EtOH (20 mL), stirred for 5 minutes and concentrated under reduced pressure. Purification by flash chromatography on silica gel eluting with 5-10% MeOH/CH₂Cl₂ gave 0.57 g (88%) of 2pA. 2pA which came from the faster eluting isomer (2p(3A)) was found to be 99.7% ee by analytical chiral normal phase chromatography. HPLC conditions: 99.7% at 2.17 min (Chiralcel OJ 44.6×250 mm, 10 micron, 40° C., isocratic 80% Heptane/20% EtOH/MeOH (1:1), 1.0 mL/min., detection at 288 nm).

2p(3B) (0.86 g, 2.19 mmol) was dissolved in 2% 12N HCl/EtOH (20 mL), stirred for 5 minutes and concentrated under reduced pressure. Purification by flash chromatography on silica gel eluting with 5-10% MeOH/CH₂Cl₂ gave 0.60 g (90%) of 2pB. 2pB which came from the slower eluting isomer (2p(3B)) was found to have 87.1% ee by analytical chiral normal phase chromatography. HPLC conditions: 87.1% at 18.4 min (Chiralcel OJ 44.6×250 mm, 10 micron, 40° C., isocratic 80% heptane/20% EtOH/MeOH (1:1), 1.0 mL/min., detection at 288 nm).

The absolute conformation for compounds 2pA and 2pB has not been determined. For simplicity in nomenclature, we have designated compound 2pA as having an “S” configuration and compound 2pB as having a “R” configuration. Enantiomerically pure products derived from compound 2pA will be designated as having a “S” configuration and enantiomerically pure products derived from compound 2pB will be designated as having a “R” configuration

Example 2q Production of [3aS-(3aα,4β,5β,7β,7aα)]-[7-[2-(4-Cyanophenoxy)ethyl]octahydro-5-hydroxy-4-methyl-1,3-dioxo-4,7-epoxy-2H-isoindol-2-yl]-1-naphthalenecarbonitrile (2qA) and [3aR-(3aα,4β,5β,7β,7aα)]-4-[7-[2-(4-Cyanophenoxy)ethyl]octahydro-5-hydroxy-4-methyl-1,3-dioxo-4,7-epoxy-2H-isoindol-2-yl]-1-naphthalenecarbonitrile, (2qB)

DBAD (26 mg, 0.115 mmol) was added to a solution of PPh₃ (30 mg, 0.115 mmol) in THF (0.65 mL). After stirring for 10 min, 4-cyanophenol (13.6 mg, 0.115 mmol) was added and the reaction mixture was stirred for a further 5 min. Compound 2pA (30 mg, 0.076 mmol) was added and the mixture was stirred at rt for 1 h. The reaction was concentrated under reduced pressure. Purification by flash chromatography on silica gel eluting with 30% acetone/70% CHCl₃ gave 23.1 mg (0.047 mmol, 61.7%) of compound 2qA. HPLC conditions: 95% at 3.06 min (YMC S5 ODS 4.6×50 mm, 10%-90% aqueous methanol over 4 minute gradient with 0.2% H₃PO₄, detecting at 220 nm). MS (ES): m/z 494.09 [M+H]⁺. [α]_(D)=53.30°, C.=4.5 mg/cc in THF, @ 589 nm)

DBAD (26 mg, 0.115 mmol) was added to a solution of PPh₃ (30 mg, 0.115 mmol) in THF (0.65 mL). After stirring for 10 min, 4-cyanophenol (13.6 mg, 0.115 mmol) was added and the reaction mixture was stirred for a further 5 min. Compound 2pB (30 mg, 0.076 mmol) was added and the mixture was stirred at rt for 1 h. The reaction was concentrated under reduced pressure. Purification by flash chromatography on silica gel eluting with 30% acetone/70% CHCl₃ gave 20.3 mg (0.041 mmol, 54.2%) of compound 2qB. HPLC conditions: 90% at 3.07 min (YMC S5 ODS 4.6×50 mm, 10%-90% aqueous methanol over 4 minute gradient with 0.2% H₃PO₄, detecting at 220 mn). MS (ES): m/z 494.09 [M+H]⁺. [α]_(D=−)42.87°, C.=6.6 mg/cc in THF, @ 589 nm)

Example 2r Production of (3aα,4β,7β,7aα)-4-[4-[2-[(6Chloro-1,2-benzisoxazol-3-yl)oxy]ethyl]octahydro-7-methyl-1,3-dioxo-4,7-epoxy-2H-isoindol-2-yl]-1-naphthalenecarbonitrile (2r)

To a solution of PPh₃ (52 mg, 0.20 mmol) in 0.5 mL THF was added DBAD (46 mg, 0.20 mmol) as one solid portion. The resulting mixture was stirred for 10 min before 6-chloro-3-hydroxy-1,2-benzisoxazole (34 mg, 0.20 mmol) was added. Stirring was continued for 10 min before a solution of 2j(2) (50 mg, 0.13 mmol) in 0.5 mL THF was introduced via canula. The resulting mixture was stirred at ambient temperature for 24 h, concentrated and purified by preparative reverse phase HPLC (YMC S5 ODS 20×100 mm column; eluting with 30-100% aqueous MeOH containing 0.1% TFA over 10 min at 20 mL/min) to yield a white solid. The obtained solids were dissolved in CH₂Cl₂, washed with sat. NaHCO₃ solution, dried over Na₂SO₄ and concentrated to yield 50 mg (71%) of 2r as a colorless oil. HPLC: 26% at 3.89 min and 74% at 4.02 min (mixture of atropisomers, retention time) (YMC S5 ODS column 4.6×50 mm Ballistic, 10-90% aqueous methanol over 4 minutes containing 0.2% H₃PO₄, 4 mL/min, monitoring at 220 nm), MS (ES): m/z 528.4 [M+H]⁺.

Example 2s Production of (3aα,4β,7β,7aα)-4-[Octahydro-4-methyl-7-[2-[(6-nitro-1H-indazol-3-yl)oxy]ethyl]-1,3-dioxo-4,7-epoxy-2H-isoindol-2-yl]-1-naphthalenecarbonitrile (2s)

To a solution of 2j(2) (50 mg, 0.13 mmol) in toluene (1 mL) was added ADDP (50 mg, 0.20 mmol), 6-nitro-3-indazolinone (36 mg, 0.20 mmol) and n-Bu₃P (50 μL, 0.2 mmol). The resulting mixture was heated to 80° C. for 24 h, concentrated and purified by a combination of preparative reverse phase HPLC (YMC S5 ODS 20×100 mm column; eluting with 30-100% aqueous MEOH containing 0.1% TFA over 10 min at 20 mL/min) and flash chromatography (silica gel, 25% acetone in CHCl₃) to give 17 mg (25%) of 2s as a yellow solid. HPLC: 24% at 3.60 min and 76% at 3.74 min (mixture of atropisomers, retention time) (YMC S5 ODS column 4.6×50 mm Ballistic, 10-90% aqueous methanol over 4 minutes containing 0.2% H₃PO₄, 4 mL/min, monitoring at 220 nm), MS (ES): m/z 537.6 [M+H]⁺.

Example 2t Production of [3aS-(3aα,4β,5β,7β,7aα)]-4-[7-[2-(1,2-Benzisoxazol-3-yloxy)ethyl]octahydro-5-hydroxy-4-methyl-1,3-dioxo-4,7-epoxy-2H-isoindol-2-yl]-1-naphthalenecarbonitrile (2t)

PPh₃ (47 mg, 0.18 mmol), DBAD (41 mg, 0.18 mmol), 3-hydroxy-1,2-benzisoxazole (24 mg, 0.18 mmol) and compound 2pA (35 mg, 0.09 mmol) were reacted according to the procedure given for 2r. Purification was achieved by reverse phase HPLC (YMC S5 ODS 20×100 mm column; eluting with 30-100% aqueous MeOH containing 0.1% TFA over 10 min at 20 mL/min) to yield a white solid. The obtained solids were dissolved in CH₂Cl₂, washed with sat. NaHCO₃ solution, dried over Na₂SO₄ and concentrated furnishing 29 mg (64%) of 2t as a colorless oil. HPLC: 96% at 3.29 min (mixture of atropisomers, retention time) (YMC S5 ODS column 4.6×50 mm Ballistic, 0-100% aqueous methanol over 4 minutes containing 0.2% H₃PO₄, 4 mL/min, monitoring at 220 nm), MS (ES): m/z 510.2 [M+H]⁺.

Example 2u Production of [3aR-(3aα,4β,5β,7β,7aα)]-4-[7-[2-(1,2-Benzisoxazol-3-yloxy)ethyl]octahydro-5-hydroxy-4-methyl-1,3-dioxo-4,7-epoxy-2H-isoindol-2-yl]-1-naphthalenecarbonitrile (2u)

PPh₃ (47 mg, 0.18 mmol), DBAD (41 mg, 0.18 mmol), 3-hydroxy-1,2-benzisoxazole (24 mg, 0.18 mmol) and 2pB (35 mg, 0.09 mmol) were reacted according to the procedure given for 2r. Purification was achieved by reverse phase HPLC (YMC S5 ODS 20×100 mm column; eluting with 30-100% aqueous MeOH containing 0.1% TFA over 10 min at 20 mL/min) to yield a white solid. The obtained solids were dissolved in CH₂Cl₂, washed with sat. NaHCO₃ solution, dried over Na₂SO₄ and concentrated furnishing 23 mg (51%) of 2u as a colorless oil. HPLC: 95% at 3.29 min (mixture of atropisomers, retention time) (YMC S5 ODS column 4.6×50 mm Ballistic, 0-100% aqueous methanol over 4 minutes containing 0.2% H₃PO₄, 4 mL/min, monitoring at 220 nm), MS (ES): m/z 510.4 [M+H]⁺.

Example 2v Production of (3aα,4β,7β,7aα)-4-[4-[2-(4-Cyanophenoxy)ethyl]-7-ethyloctahydro-1,3-dioxo-4,7-epoxy-2H-isoindol-2-yl]-1-naphthalenecarbonitrile, (2v)

2-Ethyl-5-(2-hydroxyethyl)furan (2v(1))

n-BuLi (2.5 M in hexane, 4.4 mL, 11 mmol) was added to a solution of 2-ethylfuran (1.05 mL, 10 mmol) in THY (10 mL) at −25° C. The solution was warmed to rt and stirred for 3 h. Ethylene oxide (0.75 mL) was added at −78° C. The reaction was stirred for 0.5 h at −15° C. and overnight at rt. Aqueous sat. NH₄Cl was added and the mixture was extracted with ether (3×). The combined extracts were washed with water (1×) and brine (1×) and dried over Na₂SO₄. Purification by flash chromatography on silica gel eluting with 30% EtOAc/70% hexane gave 1.12 g (8.02 mmol, 80.2%) of 2v(1) as a yellow oil.

(3aα,4β,7β,7aα)-4-[4-Ethyl-1,3,3a,4,7,7a-hexahydro-7-(2-hydroxyethyl)-1,3-dioxo-4,7-epoxy-2H-isoindol-2-yl]-1-naphthalenecarbonitrile (2v(2))

A solution of 2v(1) (280 mg, 2.00 mmol) and the 4-(2,5-dihydro-2,5-dioxo-1H-1-yl)-1-naphthalenecarbonitrile (496 mg, 2.00 mmol) in benzene (2 mL) was stirred at 60° C. for 2 h. The reaction mixture was concentrated under reduced pressure. The yellow solid, 2v(2), was used directly in the next step.

(3aα,4β,7β,7aα)-4-[4-Ethyloctahydro-7-(2-hydroxyethyl)-1,3-dioxo-4,7-epoxy-2H-isoindol-2-yl]-1-naphthalenecarbonitrile (2v(3))

A mixture of 2v(2) (764 mg, 1.97 mmol) and 10% Pd/C (115 mg, cat.) in EtOAc (36 mL) was stirred under a hydrogen atmosphere at rt for 2 h. The reaction mixture was filtered through celite and concentrated under reduced pressure to give 779 mg of crude 2v(3). Purification of this crude product by flash chromatography on silica gel eluting with 70% EtOAc/30% hexane gave 235 mg (0.6 mmol, 30.1%) of 2v(3). HPLC conditions: 99% at 2.84 min (YMC S5 ODS 4.6×50 mm, 10%-90% aqueous methanol over 4 minute gradient with 0.2% H₃PO₄, detecting at 220 nm). MS (ES): m/z 391.12 [M+H]⁺.

(3aα,4β,7β,7aα)-4-[4-[2-(4Cyanophenoxy)ethyl]-7-ethyloctahydro-1,3-dioxo-4,7-epoxy-2H-isoindol-2-yl]-1-naphthalenecarbonitrile (2v)

DBAD (44.2 mg, 0.192 mmol) was added to a solution of PPh₃ (50.4 mg, 0.192 mmol) in THF (1 mL). After stirring for 10 mins, 4-cyanophenol (23 mg, 0.192 mmol) was added and the reaction mixture was stirred for an additional 5 mins. 2v(3) (50 mg, 0.128 mmol) was added and the mixture was stirred at rt for 2 h. The reaction was concentrated under reduced pressure. Purification by flash chromatography on silica gel eluting with 40% EtOAc/60% hexane gave 43 mg (0.087 mmol, 68.4%) of compound 2v as a white solid. HPLC conditions: 99% at 3.65 min (YMC S5 ODS 4.6×50 mm, 10%-90% aqueous methanol over 4 minute gradient with 0.2% H₃PO₄, detecting at 220 nm). MS (ES): m/z 492.16 [M+H]⁺.

Example 2w Production of [3aS-(3aα,4β,5β,7β,7aα)]-4-[7-[2-(Acetyloxy)ethyl]octahydro-5-hydroxy-4-methyl-1,3-dioxo-4,7-epoxy-2H-isoindol-2-yl]-1-naphthalenecarbonitrile (2wA) and [3aR-(3aR-(3aα,4β,5β,7β,7aα)]-4-[Octahydro-5-hydroxy-7-(2-hydroxyethyl)-4-methyl-1,3-dioxo-4,7-epoxy-2H-isoindol-2-yl]-1-naphthalenecarbonitrile(2wB)

A racemic mixture of compounds 2pA and 2pB (1.90 gram) were dissolved in 100 mL of anhydrous THF in a 2 L flask. Anhydrous tert-butyl-methyl ether (900 mL) and vinyl acetate (40 mL) were transferred into the flask with stirring and lipase (20 g, type II, crude, from porcine pancreas; Sigma, Cat# L3126) was added. The reaction mixture was stirred for 21 hr at rt at which point an additional 5 grams of the lipase and 20 mL of vinyl acetate were added. The reaction was stirred at rt for an additional 19 h, stored at 4° C. without stirring for 36 h and then stirred at rt for another 22 h (until the desired % ee was apparent by chiral HPLC). To monitor the reaction, 200 uL of the mixture was withdrawn and centrifuged. The supernatant (100 uL) was dried under nitrogen and the resulting residue was dissolved in 100 uL of EtOH and subjected to HPLC analysis:

-   1) Reverse phase HPLC: Column, YMC-ODS AQ 150×4.6; flow rate, 1.2     mL/min; sample size, 10 uL

solvent A,: 1 mM HCl in water; solvent B, MeCN; monitored at 300 nm Gradient: Time(min) 0 8 8.5 9.5 10 12 B % 30 60 85 85 30 30

-   2) Chiral-HPLC: Column, CHIRALCEL OJ 4.6×250 mm     -   mobile phase, Hexane/MeOH/EtOH (8:1:1)     -   flow rate, 1 mL/min; sample size, 20 uL     -   monitored at both 220 and 300 nm     -   performed at 25° C. & 40° C.     -   (for ee % determination of reaction mixture)

The enzyme was removed by filtration and filtrate was concentrated under vacuum. The resulting mixture was dissolved in CHCl₃ and adsorbed onto silica gel (63-200 microns). These solids were applied to a VLC funnel (3 cm I.D., VLC is vacuum liquid chromatography using glass funnels having 24/40 joints at the bottom) containing a 5 cm bed height of silica gel (25-40 microns) and a step gradient was carried out. The gradient was 100% CHCl₃ in the first 3 fractions, followed by CHCl₃-1% MeOH (3 fractions), CHCl₃-2% MEOH (3 fractions), CHCl₃-3% MeOH (3 fractions), CHCl₃-4% MEOH (3 fractions), and finally with CHCl₃-5% MeOH (3 fractions). The volume of the fractions was 100 mL until reaching CHCl₃-3% MEOH and from that point on it was 200 mL. 2wA elutes in the last two fractions of 100% CHCl₃ and until the first fraction of CHCI₃-2% MEOH. 2wB elutes starting with the second fraction of CHCl₃-2% MEOH, and continues to the first fraction of CHCl₃-5% MeOH. The crude compound 2wB contained a small amount of a colored impurity which was removed by a Sephadex column [LH-20 swollen in CHCl₃—MeOH (2:1), column (2.5 cm I.D. & 90 cm long) to yield 632 mg of compound 2wB. Compound 2wA: HPLC conditions: 98% at 7.2 min (method 1), chiral HPLC conditions: 29.0 min @ 25° C. (method 2). Compound 2wB: HPLC conditions: 98% at 4.6 min (method 1), chiral HPLC conditions: 96% ee at 25.7 min (@ 25° C.) & 19.8 min @ 40° C.) (method 2).

Example 2x Production of (3aα,4β,7β,7aα)-4-[4-[2-[[(1,1-Dimethylethyl)dimethylsilyl]oxy]ethyl]-1,3,3a,4,7,7a-hexahydro-7-methyl-1,3-dioxo-4,7-epoxy-2H-isoindol-2-yl]-2-(trifluoromethyl)benzonitrile & (3aα,4α,7α,7aα)-4-4-[4-(2-[[(1,1-Dimethylethyl)dimethylsilyl]oxy]ethyl]-1,3,3a,4,7,7a-hexahydro-7-methyl-1,3-dioxo-4,7-epoxy-2H-isoindol-2-yl]-2-(trifluoromethyl)benzonitrile (2xA & 2xB)

Compound 2h(2) (2.00 g, 8.50 mmol) and 4-(2,5-Dihydro-2,5-dioxo-1H-5 pyrrol-1-yl)-2-trifluoromethylbenzonitrile (1.50 g, 5.60 mmol) were mixed in benzene (5.0 mL) and heated at 60° C. for 14 h, then cooled to 25° C. The solvent was removed at 40° C. under vacuum for 1 h to give the crude material which was purified by flash chromatography on SiO₂ eluting with 0.5% EtOAc/CH₂Cl₂ to give 2.0 g of compound 2xA and 1.3 g of compound 2xB, both as light brown solids. Compound 2xA: HPLC: 95% at 4.200 min (retention time) (YMC S5 ODS column 4.6×50 mm eluting with 10-90% aqueous methanol over 4 minutes containing 0.2% phosphoric acid, 4 mL/min, monitoring at 220 mn), MS (ES): m/z 507.1 [M+H]⁺. Compound 2xB: HPLC: 95% at 4.20 min (retention time) (YMC S5 ODS column 4.6×50 mm eluting with 10-90% aqueous methanol over 4 minutes containing 0.2% phosphoric acid, 4 mL/min, monitoring at 220 nm), MS (ES): m/z 507.1 [M+H]⁺.

Example 2y Production of [3aR-(3aα,4β,5β,7β,7aα)]-4-[7-[2-[[(1,1-Dimethylethyl)dimethylsilyl]oxy]ethyl]octahydro-5-hydroxy-4-methyl-1,3-dioxo-4,7-epoxy-2H-isoindol-2-yl]-2-(trifluoromethyl)benzonitrile & [3aS-(3aα,4β,5β,7β,7aα)-4-[7-[2-[[(1,1-Dimethylethyl)dimethylsilyl]oxy]ethyl]octahydro-5-hydroxy-4-methyl-1,3-dioxo-4,7-epoxy-2H-isoindol-2-yl]-2-(trifluoromethyl)benzonitrile (2yA & 2yB)

Compound 2xA (1.40 g, 2.77 mmol) and RhCl(PPh₃)₃ (0.128 g, 0.14 mmol) were mixed in a flask. The flask was then evacuated and filled with argon three times, followed by the syringe addition of THF (3.0 mL). Once all particulates were dissolved, catecholborane (0.59 mL, 5.54 mmol) was added dropwise. The reaction mixture was stirred at 25° C. under argon for 30 min, then cooled to 0° C. Phosphate buffer (pH=7, 20 mL) was added, followed by EtOH (10 mL), 30% H₂O₂/H₂O (2 mL). The reaction mixture was stirred at 0° C. for 3 h, then extracted with dichloromethane (3×25 mL). The combined organic layers were washed with 1 N NaOH (25 mL), 10% Na₂SO₃ (25 mL) and brine (25 mL). The crude material was then concentrated and purified by flash chromatography on SiO₂ eluting with 2% EtOAc/CH₂Cl₂ to 10% EtOAc/CH₂Cl₂ to give 0.63 g of a racemic mixture of compounds 2yA & 2yB as a light yellow solid. HPLC: 99% at 3.867 min (retention time) (YMC S5 ODS column 4.6×50 mm eluting with 10-90% aqueous methanol over 4 minutes containing 0.2% phosphoric acid, 4 mL/min, monitoring at 220 nm), MS (ES): m/z 525.1 [M+H]

The racemic mixture of compounds 2yA & 2yB was separated by normal phase preparative chiral HPLC using a Chiracel OD column (5 cm×50 cm), eluting with 13% solvent B (EtOH) in solvent A (Hexane), flow rate: 50 mL/min. Compound 2yA eluted from 34 min to 38 min and compound 2yB eluted from 44 min to 49 min. Enantiomeric excess was determined by chiral HPLC. Compound 2yA: >99% ee (12.576 min (retention time) (Chiralcel OJ column 4.6×250 mm eluting with isocratic 85% heptane/15% MeOH/ethanol (1:1), 1 mL/min, monitoring at 220 nm, 40° C.). Compound 2yB: 99% ee (18.133 min (retention time) (Chiralcel OJ column 4.6×250 mm eluting with isocratic 85% heptane/15% MeOH/ethanol (1:1), 1 mL/min, monitoring at 220 nm, 40° C.).

The absolute configuration for compounds 2yA & 2yB were not established. For simplicity in nomenclature, compound 2yA is designated herein as having an “R” configuration and compound 2yB as having an “S” configuration. Enantiomerically pure products derived from compound 2yA are designated herein as having a “R” configuration and enantiomerically pure products derived from compound 2yB are designated herein as having an “S” configuration.

Example 2z Production of [3aR-(3aα,4β,5β,7β,7aα)]-4-[Octahydro-5-hydroxy-7-(2-hydroxyethyl)-4-methyl-1,3dioxo-4,7-epoxy-2H-isoindol-2-yl]-2-(trifluoromethyl)benzonitrile & [3aS-(3aα,4β,5β,7β,7aα)]-4-[Octahydro-5-hydroxy-7-(2-hydroxyethyl)-4methyl-1,3-dioxo-4,7epoxy-2H-isoindol-2-yl]-2-(trifiuoromethyl)benzonitrile (2zA & 2zB)

Compound 2yA (180 mg, 0.34 mmol) was dissolved in 2% HCl/EtOH (5.0 mL). After 30 min, saturated NaHCO₃ was added and the aqueous layer was extracted with dichloromethane (20 mL×3), washed with brine and dried over Na₂SO₄ to give 135 mg of compound 2zA as a white solid. HPLC: 99% at 2.257 min (retention time) (YMC S5 ODS column 4.6×50 mm eluting with 10-90% aqueous methanol over 4 minutes containing 0.2% phosphoric acid, 4 mL/min, monitoring at 220 nm), MS (ES): m/z 411.1 [M+H]⁺.

The above procedure was repeated with compound 2yB to yield the desired diol compound 2zB in similar yield.

Example 2a(i) Production of [3aR-(3aα,4β,5β,7β,7aα)]-4-[7-[2-[(5-Chloro-2-pyridinyl)oxy]ethyl]octahydro-5-hydroxy-4-methyl-1,3-dioxo-4,7-epoxy-2H-isoindol-2-yl]-2-(trifluoromethyl)benzonitrile (2a(i))

Triphenylphosphine (0.026 g, 0.098 mmol) and DBAD (0.023 g, 0.098 mmol) were mixed in THF (0.5 mL). After allowing the previous mixture to react for 15 min, 2-hydroxy-6-chloropyridine (0.016 g, 0.100 mmol) was added, the mixture was allowed to stir for 10 min and compound 2zA (0.020 g, 0.049 mmol) was added. The reaction mixture was stirred at 25° C. for 2 h and then the crude material was purified by preparative TLC, eluting with 10% acetone/CHCl₃, to give 0.014 g of compound 2a(i) as a light brown solid. HPLC: 100% at 3.370 min (retention time) (YMC S5 ODS column 4.6×50 mm eluting with 10-90% aqueous methanol over 4 minutes containing 0.1% TFA, 4 mL/min, monitoring at 220 nm), MS (ES): m/z 522.08 [M+H]⁺.

Example 2b(i) Production of (3aα,4β,5β,7β,7aα)-4-[4-Ethyloctahydro-5-hydroxy-7-(2-hydroxyethyl)-1,3-dioxo-4,7-epoxy-2H-isoindol-2-yl]-1-naphthalenecarbonitrile (2b(i)C)

tert-Butyl-[2-(5-ethyl-furan-2-yl)-ethoxy]-dimethyl-silane (2b(i)A)

Imidazole (255 mg, 3.75 mmol) and TBSCl (414 mg, 2.75 mmol) were added to the solution of (2v(1)) (350 mg, 2.5 mmol) in DMF (4 mL). The mixture was stirred at rt for 15 hr and then 100 mg (0.66 mmol) of additional TBSCl was added to drive the reaction to completion. After stirring for an additional hour, the reaction mixture was diluted with diethylether (100 mL) and washed with water (20 mL), 1 N HCl (20 mL), water (20 mL) and brine (20 mL). The organic layer was dried over Na₂SO₄ and concentrated under reduced pressure to give 509 mg of compound 2b(i)A (80.3%) as a yellow oil.

(3aα,4β,7β,7aα)-4-[4-[2-[[(1,1-Dimethylethyl)dimethylsilyl]oxy]ethyl]-4-ethyl-1,3,3a,4,7,7a-hexahydro-1,3-dioxo-4,7-epoxy-2H-isoindol-2-yl]-1-naphthalenecarbonitrile (2b(i)B)

A solution of compound 2b(i)A (509 mg, 2.00 mmol) and 4-(2,5-dihydro-2,5-dioxo-1H-1-yl)-1-naphthalenecarbonitrile (498 mg, 2.00 mmol) in benzene (2 mL) was heated at 60° C. for 18 h. The reaction mixture was concentrated under reduced pressure to give 992 mg (99%) of crude compound 2b(i)B, which was used directly in the next step without further purification.

(3aα,4β,5β,7β,7aα)-4-[4-Ethyloctahydro-5-hydroxy-7-(2-hydroxyethyl)-1,3-dioxo-4,7-epoxy-2H-isoindol-2-yl]-1-naphthalenecarbonitrile (2b(i)C)

A mixture of compound 2b(i)B (992 mg, 1.98 mmol) and RhCl₂(PPh₃)₃ (183 mg, 0.198 mmol) was evacuated and filled with argon (3×). THF (20 mL) was added and once all particulates had dissolved, catecholborane (0.42 mL, 3.96 mmol) was slowly added dropwise. When the formation of product ceased, as was determined by HPLC, the reaction mixture was cooled to 0° C. and quenched with phosphate buffer (34 mL, pH 7.2) followed by the addition of EtOH (19 mL) and H₂O₂ (2.9 mL, 30% aq sol). After 2 h, additional phosphate buffer (6.8 mL, pH 7.2), EtOH (3.8 mL) and H₂O₂ (0.6 mL) were added. The reaction mixture was stirred at rt for 3 h. Once the boronate intermediate was consumed, the mixture was extracted with CH₂Cl₂ (300 mL) and the combined organic layers were washed with 1N NaOH, 10% aq NaHSO₃ and brine. The combined organic layers were dried over Na₂SO₄. Purification by flash chromatography on silica gel eluting with 10% MeOH/CH₂Cl₂ gave 75 mg (9.3%) of compound 2b(i)C as a gray solid. HPLC conditions: 97% at 2.43 min (Phenomenex-prime S5-C18 column 4.6×50 mm, 10%-90% aqueous methanol over 4 minute gradient with 0.2% H₃PO₄, detecting at 220 nm). MS (ES): m/z 407.18 [M+H]⁺.

Example 2c(i) Production of (3aα,4β,5β,7β,7aα)-4-[7-[2-(4-Cyanophenoxy)ethyl]-4-ethyloctahydro-5-hydroxy-1,3-dioxo-4,7-epoxy-2H-isoindol-2-yl]-1-naphthalenecarbonitrile (2c(i))

DBAD (39.6 mg, 0.172 mmol) was added to a solution of PPh₃ (45.1 mg, 0.172 mmol) in THF (0.8 mL). After stirring for 10 min, 4-cyanophenol (20.5 mg, 0.172 mmol) was added and the reaction mixture was stirred for an additional 5 min Compound 2b(i)C (25.0 mg, 0.062 mmol) was added and the mixture was stirred at rt for 2 h. The reaction was concentrated under reduced pressure. Purification by Prep TLC eluting with 10% acetone/CHCl₃ gave 18.1 mg (0.036 mmol, 57.6%) of compound 2c(i). HPLC conditions: 96% at 3.15 min (YMC S5 ODS 4.6×50 mm, 10%-90% aqueous methanol over 4 minute gradient with 0.2% H₃PO₄, detecting at 220 nm). MS (ES): m/z 508.14 [M+H]⁺.

Example 2d(i) Production of (3aα,4β,5β,7β,7aα)-4-[Octahydro-5-hydroxy-7-(2-hydroxyethyl)-4-methyl-1,3dioxo-4,7-epoxy-2H-isoindol-2-yl]-1-naphthalenecarbonitrile (2d(i))

Compounds (2j(1)) and (2j(2)) were converted to compound 2d(i) by biotransformation.

Microbial Hydroxylation of Compound 2j(1)

Step 1: Reaction

One frozen vial (approximately 2 ml) of Streptomyces griseus ATCC 10137 was added to a 500 ml flask containing 100 ml of transformation medium. The transformation medium was prepared as follows: to a 2 liter plastic beaker, 20 g of dextrose, 5.0 g of yeast extract, 5.0 g of soybean meal, 5.0 g of sodium chloride, 5.0 g of potassium phosphate, diabasic and one liter of deionized water were added and the mixture was stirred at room temperature for 3 to 30 min. The pH of the mixture was then adjusted to 7.0 with 1 N HCl or 1 N NaOH. The resulting mixture was dispensed into 500 ml flask (100 ml per flask). The flasks were covered with Bio/Wrap and autoclaved at 121° C. for 15 min. and cooled down to room temperature before use.

The culture was incubated at 28° C. and 250 rpm for 3 days. One ml of the resulting culture was added to a 500 ml flask containing 100 ml of the transformation medium and the flask was incubated at 28° C. and 250 rpm for 24 hours. Ten ml of the resulting culture was transferred to a 50 ml flask, to which 1 mg of compound 2j(1) in 0.2 ml ethanol was added. The flask was incubated at 28° C. and 250 rpm for 23 hours and the reaction culture was extracted with EtOAc (10 ml). The EtOAc extract was dried under N₂ and the residue was dissolved in 1 ml of MEOH (reaction extract). HPLC analysis showed that peak area ratio of compound 2d(i) to compound 2j(1) in the reaction culture was about 1.1/1.

Step 2: Product Analysis

HPLC:

10 μl of the reaction extract was injected into HPLC column (YMC ODS-AQ C-18 column, 150×6.0 mm i.d.). The column was eluted with 1 mM HCl in water/CH₃CN at 1.2 ml/min flow rate: 30 to 60% CH₃CN over 8 min, 60 to 85% CH₃CN over 0.5 min. 85% CH₃CN for 1 min, 85 to 30% CH₃CN over 0.5 min. The eluents were monitored at 300 nm. Two major peaks with about 1 to 1 area ratio were observed, which had same UV spectra as those of compounds 2d(i) and (2j(1)), and had retention times of 4.55 min and 7.23 min, respectively, matching the retention times of authentic samples of compound 2d(i)(4.53 min) and compound (2j(1)) (7.2 min).

LC/MS

The reaction extract: two major UV peaks.

-   Peak 1, Tr 4.68 min: 391 [M+H]⁺, 343, 319, 303, 289 -   Peak 2, Tr 5.35 min: 375 [M+H]⁺, 345     Authentic Samples -   Compound 2d(i), Tr 4.82 min: 391 [M+H]⁺, 343, 319, 289 -   Compound (2j(1)), Tr 5.48 min: 375 [M+H]⁺, 345

As will be understood by those of skill in the art upon reading this disclosure, additional SARMs for use in the present invention can also be identified in accordance with the methods described herein. For example, a test compound suspected of having selective androgen receptor modulating activity can be screened for antagonist activity in a hormone-dependent tumor cell line such as a human or mouse breast tumor cell line and screened for agonist activity in another nontumor androgen receptor containing cell line such as a muscle, prostate or seminal vesicle cell line as described in the assays below. These screening assays can be performed routinely in accordance with the teachings provided herein.

Example 3 AR Binding Assay

For the whole cell binding assay, human LNCaP cells (T877A mutant AR) or MDA 453 (wild type AR) in 96-well microtiter plates containing RPMI 1640 or DMEM supplemented with 10% charcoal stripped CA-FBS (Cocaleco Biologicals), respectively, were incubated at 37° C. to remove any endogenous ligand that might be complexed with the receptor in the cells. After 48 hours, either a saturation analysis to determine the K_(d) for tritiated dihydrotestosterone, [³H]-DHT, or a competitive binding assay to evaluate the ability of test compounds to compete with [³H]-DHT were performed. For the saturation analysis, media (RPMI 1640 or DMEM—0.2% CA-FBS) containing [³H]-DHT (in concentrations ranging from 0.1 nM to 16 nM) in the absence (total binding) or presence (non-specific binding) of a 500-fold molar excess of unlabeled DHT were added to the cells. After 4 hours at 37° C., an aliquot of the total binding media at each concentration of [³H]-DHT was removed to estimate the amount of free [³H]-DHT. The remaining media was removed, cells were washed three times with PBS and harvested onto UniFilter GF/B plates (Packard), Microscint (Packard) was added and plates counted in a Top-Counter (Packard) to evaluate the amount of bound [³H]-DHT.

For the saturation analysis, the difference between the total binding and the non-specific binding was defined as specific binding. The specific binding was evaluated by Scatchard analysis to determine the K_(d) for [³H]-DHT. See e.g. D. Rodbard, Mathematics and statistics of ligand assays: an illustrated guide: In: J. Langon and J. J. Clapp, eds., Ligand Assay, Masson Publishing U.S.A., Inc., New York, pp. 45-99, (1981), the disclosure of which is herein incorporated by reference.

For the competition studies, media containing 1 nM [³H]-DHT and a test compound in concentrations ranging from 10⁻¹⁰ to 10⁻⁵ M were added to the cells.

Two replicates were used for each sample. After 4 hours at 37° C., cells were washed, harvested and counted as described above. The data was plotted as the amount of [³H]-DHT (% of control in the absence of test compound) remaining over the range of the dose response curve for a given compound. The concentration of test compound that inhibited 50% of the amount of [³H]-DHT bound in the absence of competing ligand was quantified (IC₅₀) after log-logit transformation. The K_(i) values were determined by application of the Cheng-Prusoff equation to the IC₅₀ values, where: $K_{I} = {\frac{{IC}_{50}}{\left( {1 + {{\left( {}^{3}{H\text{-}{DHT}} \right)/K_{d}}\quad{for}\quad{\,^{3}H}\text{-}{DHT}}} \right)}.}$ After correcting for non-specific binding, IC₅₀ values were determined. The IC₅₀ is defined as the concentration of competing ligand needed to reduce specific binding by 50%. The K_(d)s for [³ H]-DHT for MDA 453 and LNCaP were 0.7 and 0.2 nM respectively.

Example 4 Human Prostate Cell Proliferation Assay

The effects of test compound on proliferation of human prostate cancer cell lines was also examined. For that, MDA PCa2b cells, a cell line derived from the metastasis of a patient that failed castration (Navone et al., Clin. Cancer Res., 3, 2493-10 500 (1997)), were incubated with or without the test compounds for 72 hours and the amount of [³H]-thymidine incorporated into DNA was quantified as a way to assess number of cells and therefore proliferation. The MDA PCa2b cell line was maintained in BRFF-HPCl media (Biological Research Faculty & Facility Inc., MD) supplemented with 10% FBS. For the assay, cells were plated in Biocoated 96-well microplates and incubated at 37° C. in 10% FBS (charcoal-stripped)/BRFF-BMZERO (without androgens). After 24 hours, the cells were treated in the absence (blank) or presence of 1 nM DHT (control) or with test compounds (sample) in concentrations ranging from 10⁻¹⁰ to 10⁻⁵M. Duplicates were used for each sample. The compound dilutions were performed on a Biomek 2000 laboratory work station. Seventy two hours later 0.44 uCi. of [³H]-Thymidine (Amersham) was added per well and incubated for another 24 h followed by trypsinization, harvesting of the cells onto GF/B filters. Micro-scint PS were added to the filters before counting them on a Beckman TopCount.

The % Inhibition was calculated as: % Inhibition=100×(1−[average_(control)−average_(blank)/average_(sample)−average_(blank)]) Data was plotted and the concentration of compound that inhibited 50% of the [³H]-Thymidine incorporation was quantified (IC₅₀).

Example 5 C2C12 Mouse Myoblast Transactivation Assay

Two functional transactivation assays were developed to assess the efficacy of androgen agonists in a muscle cell background using a luciferase reporter. The first assay (ARTA Stable 1) uses a cell line, Stable 1 (clone #72), which stably expresses the full length rat androgen receptor but requires the transient transfection of an enhancer/reporter. This cell line was derived from C2C 12 mouse myoblast cells. The second assay (ARTA Stable 2) uses a cell line, Stable 2 (clone #133), derived from Stable 1, which stably expresses both rAR and the enhancer/luciferase reporter. These assays and cell lines

The enhancer/reporter construct used in this system is pGL3/2XDR-1/luciferase. 2XDR-1 was reported to be an AR specific response element in CV-1 cells, Brown et. al. The Journal of Biological Chemistry 272, 8227-8235, (1997). It was developed by random mutagenesis of an AR/GR consensus enhancer sequence.

For the ARTA Stable 1 assay, Stable 1 cells are plated in 96 well format at 6,000 cells/well in high glucose DMEM without phenol red (Gibco BRL, Cat. No.: 21063-029) containing 10% charcoal and dextran treated FBS (Hyclone Cat. No.: SH30068.02), 50 mM HEPES Buffer (Gibco BRL, Cat. No.: 15630-080), 1× MEM Na Pyruvate (Gibco BRL, Cat. No.: 11360-070), 0.5× Antibiotic-Antimycotic, and 800 ug/ml Geneticin (Gibco BRL, Cat. No.: 10131-035). Forty-eight hours later, cells are transfected with pGL3/2XDR-1/luciferase using LipofectAMINE Plus™ Reagent (Gibco BRL, Cat. No.: 10964-013). Specifically, 5 ng/well pGL3/2XDR-1/luciferase DNA and 50 ng/well Salmon Sperm DNA (as carrier) are diluted with 5 μl/well Opti-MEMem media (Gibco BRL, Cat. No.: 31985-070). To this, 0.5 μl/well Plus reagent is added. This mixture is incubated for 15 minutes at room temperature. In a separate vessel, 0.385 ul/well LipofectAMINE reagent is diluted with 5 μl/well Opti-MEM. The DNA mixture is then combined with the LipofectAMINE mixture and incubated for an additional 15 minutes at room temperature. During this time, the media from the cells is removed and replaced with 60 μl/well of Opti-MEM. To this is added 10 μl/well of the DNA/LipofectAMINE transfection mixture. The cells are incubated for 4 hours. Following the incubation, the transfection mixture is removed from the cells and replaced with 90 ul of high glucose DMEM without phenol red (Gibco BRL, Cat. No.: 21063-029) containing 10% charcoal and dextran treated FBS (HyClone Cat. No.: SH30068.02), 50 mM HEPES Buffer (Gibco BRL, Cat. No.: 15630-080), 1× MEM Na Pyruvate (Gibco BRL, Cat. No.: 11360-070), 0.5× Antibiotic-Antimycotic, and 800 μg/ml Geneticin (Gibco BRL, Cat. No.: 10131-035). Test compounds, 10 μl/well at an appropriate drug dilution, are then placed in each well. Twenty-four hours later, the Steady-Glo™ Luciferase Assay System is used to detect activity according to the manufacturer's instructions (Promega, Cat. No.: E2520).

For the ARTA stable 2 assay, Stable 2 cells are plated in 96 well format at 6,000 cells/well in high glucose DMEM without phenol red (Gibco BRL, Cat. No.: 21063-029) containing 10% charcoal and dextran treated FBS (HyClone Cat. No.: SH30068.02), 50 mM HEPES Buffer (Gibco BRL, Cat. No.: 15630-080), 1× MEM Na Pyruvate (Gibco BRL, Cat. No.: 11360-070), 0.5× Antibiotic-Antimycotic, 800 μg/ml Geneticin (Gibco BRL, Cat. No.: 10131-035) and 800 μg/ml Hygromycin β (Gibco BRL, Cat. No.: 10687-010). Forty-eight hours later, the media on the cells is removed and replaced with 90 μl fresh. Test compounds, 10 μl/well at an appropriate drug dilution, are then placed in each well. Twenty-four hours later, the Steady-Glo™ Luciferase Assay System is used to detect activity according to the manufacturer's instructions (Promega, Cat No.: E2520). See U.S. patent application Ser. No.______ (unassigned), entitled “Cell Lines and Cell-Based Assays for Identification of Androgen Receptor Modulators”, filed Jun. 20, 2001, by Jacek Ostrowski et al. (Attorney Docket No. D0177), which Patent Application is incorporated herein by reference by its entirety.

Example 6 Murine Breast Cell Proliferation Assay

The ability of test compounds to modulate the function of the AR was determined by testing said compounds in a proliferation assay using the androgen responsive murine breast cell line derived form the Shionogi tumor (Hiraoka et al., Cancer Res., 47, 6560-6564 (1987)). Stable AR dependent clones of the parental Shionogi line were established by passing tumor fragments under the general procedures originally described in Tetuo, et al. (Cancer Research 25, 1168-1175 (1965)). From the above procedure, one stable line, SC114, was isolated, characterized and utilized for the testing of example compounds. SC114 cells were incubated with or without the test compounds for 72 hours and the amount of [3H]-thymidine incorporated into DNA was quantified as a surrogate endpoint to assess the number of cells and therefore the proliferation rate as described in Suzuki et. al. (J. Steroid Biochem. Mol. Biol. 37, 559-567 (1990)). The SC114 cell line was maintained in MEM containing 10⁻⁸ M testosterone and 2% DCC-treated FCS. For the assay, cells were plated in 96-well microplates in the maintenance media and incubated at 37° C. On the following day, the medium was changed to serum free medium [Ham's F-12:MEM (1;1, v/v) containing 0.1% BSA] with (antagonist mode) or without (agonist mode) 10⁻⁸ M testosterone and the test compounds of the present invention in concentrations ranging from 10⁻¹⁰ to 10⁻⁵ M. Duplicates were used for each sample. The compound dilutions were performed on a Biomek 2000 laboratory work station. Seventy two hours later 0.44 uCi. of [3H]-Thymidine (Amersham) was added per well and incubated for another 2 hr followed by trypsinization, and harvesting of the cells onto GF/B filters. Micro-scint PS were added to the filters before counting them on a Beckman TopCount.

For the antagonist mode, the % Inhibition was calculated as: % Inhibition=100×(1−[average_(sample)−average_(blank)/average_(control)−average_(blank])) Data was plotted and the concentration of compound that inhibited 50% of the [³H]-Thymidine incorporation was quantified (IC₅₀).

For the agonist mode, % Control was referred as the effect of the tested compound compared to the maximal effect observed with the natural hormone, in this case DHT, and was calculated as: % Control=100×(average_(sample)−average_(blank))/(average_(control)−average_(blank)) Data was plotted and the concentration of compound that inhibited 50% of the [³H]-Thymidine incorporation was quantified (EC₅₀).

Example 7 Wet Prostate Weight Assay AR Antagonist Assay

The activity of test compounds as AR antagonists was investigated in an immature male rat model, a standard, recognized test of antiandrogen activity of a given compound (Hershberger et al. Proc. Soc. Expt. Biol. Med., 83, 175 (1953);

Walsh. P. C. and Gittes, R. F., Endocrinology, 86, 624 (1970); and Furr et al., J. Endocrinol., 113, R7-9 (1987)). The basis of this assay is the fact that male sexual accessory organs, such as the prostate and seminal vesicles, play an important role in reproductive function. These glands are stimulated to grow and are maintained in size and secretory function by the continued presence of serum testosterone (T), which is the major serum androgen (>95%) produced by the Leydig cells in the testis under the control of the pituitary luteinizing hormone (LH) and follicle stimulating hormone (FSH). Testosterone is converted to the more active form, dihydrotestosterone, (DHT), within the prostate by 5α-reductase. Adrenal androgens also contribute about 20% of total DHT in the rat prostate, compared to 40% of that in 65-year-old men (Labrie et al. Clin. Invest. Med., 16, 475-492 (1993)). However, this is not a major pathway, since in both animals and humans castration leads to almost complete involution of the prostate and seminal vesicles without concomitant adrenalectomy. Therefore, under normal conditions, the adrenals do not support significant growth of prostate tissues (Luke, M. C. and Coffey, D. S. “The Physiology of Reproduction” ed. By E. Knobil and J. D. Neill, 1, 1435-1487 (1994)). Since the male sex organs are the tissues most responsive to modulation of the androgen activity, this model is used to determine the androgen dependent growth of the sex accessory organs in immature castrated rats.

Male immature rats (19-20 days old Sprague-Dawley, Harlan Sprague-Dawley) were castrated under metofane anesthesia Five days after surgery these castrated rats (60-70 g, 23-25 day-old) were dosed for 31 days. Animals were dosed sub-cutaneously (s.c.) 1 mg/kg with Testosterone Proprionate (TP) in arachis oil vehicle and test compounds (compounds of the present invention) were administered orally by gavage (p.o.) in dissolved/suspensions of 80% PEG 400 and 20% Tween 80 (PEGTW). Animals were dosed (v/w) at 0.5 ml of vehicle /100 g body weight. Experimental groups were as follows:

-   -   1. Control vehicle     -   2. Testosterone Propionate (TP) (3 mg/rat/day, subcutaneous)     -   3. TP plus Casodex (administered p.o. in PEGTW, QD), a         recognized antiandrogen, as a reference compound.     -   4. To assess antagonist activity, a test compound was         administered (p.o. in PEGTW, QD) with TP (s.c. as administered         in group 2) in a range of doses.     -   5. To assess agonist activity, a test compound was administered         alone (p.o. in PEGTW, QD) in a range of doses.

At the end of the 3-day treatment, the animals were sacrificed, and the ventral prostate weighed. To compare data from different experiments, weights of the sexual organs were first standardized as mg per 100 g of body weight, and the increase in organ weight induced by TP was considered as the maximum increase (100%). ANOVA followed by one-tailed Student or Fischer's exact test was used for statistical analysis.

The gain and loss of sexual organ weight reflect the changes of the cell number (DNA content) and cell mass (protein content), depending upon the serum androgen concentration (Okuda et al., J. Urol., 145, 188-191 (1991)). Therefore, measurement of organ wet weight is sufficient to indicate the bioactivity of androgens and androgen antagonists. In immature castrated rats, replacement of exogenous androgens increases seminal vesicles (SV) and the ventral prostate (VP) in a dose dependent manner.

The maximum increase in organ weight was 4 to 5-fold when dosing 3 mg/rat/day of testosterone (T) or 1 mg/rat/day of testosterone propionate (TP) for 3 days. The EC₅₀ of T and TP were about 1 mg and 0.03 mg, respectively. The increase in the weight of the VP and SV also correlated with the increase in the serum T and DHT concentration. Although administration of T showed 5-times higher serum concentrations of T and DHT at 2 hours after subcutaneous injection than that of TP, thereafter, these high levels declined very rapidly. In contrast, the serum concentrations of T and DHT in TP-treated animals were fairly consistent during the 24 hours, and therefore, TP showed about 10-30-fold higher potency than free T.

In this immature castrated rat model, a known AR antagonist (Casodex) was also administered simultaneously with 0. 1 mg of TP (ED₈₀), inhibiting the testosterone-mediated increase in the weights of the VP and SV in a dose dependent manner. The antagonist effects were similar when dosing orally or subcutaneously. SARMs of the invention also exhibited AR antagonist activity by suppressing the testosterone-mediated increase in the weights of VP and SV.

Example 8 Levator Ani & Wet Prostate Weight Assay AR Agonist Assay

The activity of test compounds as AR agonists was investigated in an immature male rat model, a recognized test of anabolic effects in muscle and sustaining effects in sex organs for a given compound (Hershberger et al., Proc. Soc. Expt. Biol. Med., 83, 175 (1953); Beyler et al., J. Amer. Med. Women's Ass., 23, 708 (1968); Fukuda et al., Nago Dai. Yak Ken. Nem. 14, 84 (1966)). The basis of this assay lies in the well-defined action of androgenic agents on the maintenance and growth of muscle tissues and sexual accessory organs in animals and man. Androgenic steroids, such as testosterone (T), have been well characterized for their ability to maintain muscle mass. Treatment of animals or humans after castrations with an exogenous source of T results in a reversal of muscular atrophy. The effects of T on muscular atrophy in the rat levator ani muscle have been well characterized (Masuoka et al., Am. J. Anat. 119, 263 (1966); Gori et al., Boll.—Soc. Ital. Biol. Sper. 42, 1596 (1966); Gori et al., Boll.—Soc. Ital. Biol. Sper. 42, 1600 (1966); Boris et al., Steroids 15, 61 (1970)). As described in Example 6, the effects of androgens on maintenance of male sexual accessory organs, such as the prostate and seminal vesicles, is well described. Castration results in rapid involution and atrophy of the prostate and seminal vesicles. This effect can be reversed by exogenous addition of androgens. Since both the levator ani muscle and the male sex organs are the tissues most responsive to the effects of androgenic agents, this model is used to determine the androgen dependent reversal of atrophy in the levator ani muscle and the sex accessory organs in immature castrated rats.

Sexually mature rats (200-250 g, 6-8 weeks-old, Sprague-Dawley, Harlan) were acquired castrated from the vendor (Taconic). The rats were divided into groups and treated daily for 7 to 14 days with one of the following:

-   -   1. Control vehicle     -   2. Testosterone Propionate (TP) (3 mg/rat/day, subcutaneous)     -   3. TP plus Casodex (administered p.o. in PEGTW, QD) , a         recognized antiandrogen, as a reference compound.     -   4. To assess antagonist activity, a test compound was         administered (p.o. in PEGTW, QD) with TP (s.c. as administered         in group 2) in a range of doses.     -   5. To assess agonist activity, a test compound was administered         alone (p.o. in PEGTW, QD) in a range of doses.

At the end of the 7-14-day treatment, the animals were sacrificed by carbon dioxide, and the levator ani, seminal vesicle and ventral prostate weighed. To compare data from different experiments, the levator ani muscle and sexual organ weights were first standardized as mg per 100 g of body weight, and the increase in organ weight induced by TP was considered as the maximum increase (100%). Super-anova (one factor) was used for statistical analysis.

The gain and loss of sexual organ weight reflect the changes of the cell number (DNA content) and cell mass (protein content), depending upon the serum androgen concentration (Okuda et al., J. Urol., 145, 188-191 (1991)). Therefore, measurement of organ wet weight is sufficient to indicate the bioactivity of androgens and androgen antagonist. In immature castrated rats, replacement of exogenous androgens increases levator ani, seminal vesicles (SV) and prostate in a dose dependent manner.

The maximum increase in organ weight was 4- to 5-fold when dosing 3 mg/rat/day of testosterone (T) or 1 mg/rat/day of testosterone propionate (TP) for 3 days. The EC₅₀ of T and TP were about 1 mg and 0.03 mg, respectively. The increase in the weight of the VP and SV also correlated with the increase in the serum T and DHT concentration. Although administration of T showed 5-times higher serum concentrations of T and DHT at 2 hours after subcutaneous injection than that of TP, thereafter, these high levels declined very rapidly. In contrast, the serum concentrations of T and DHT in TP-treated animals were fairly consistent during the 24 hours, and therefore, TP showed about 10-30-fold higher potency than free T.

Example 9 Mature Rat Prostate Weight Assay

The activity of test compounds was also investigated in a mature male rat model, which is a variation of the Levator ani and wet prostate weight assay described in Example 7. The in vivo assays of Examples 6 and 7 are recognized assays for determining the anabolic effects in muscle and sustaining effects in sex organs for a given compound (Hershberger et al., Proc. Soc. Expt. Biol. Med., 83, 175 (1953); Beyler et al., J. Amer. Med. Women's Ass. 23, 708 (1968); Fukuda et al., Nago Dai. Yak. Ken. Nem. 14, 84 (1966)). The basis of this assay lies in the well-defined action of androgenic agents on the maintenance and growth of muscle tissues and sexual accessory organs in animals and man.

The male sexual accessory organs, such as the prostate and seminal vesicles, play an important role in reproductive function. These glands are stimulated to grow and are maintained in size and secretory function by the continued presence of serum testosterone (T), which is the major serum androgen (>95%) produced by the Leydig cells in the testis under the control of the pituitary luteinizing hormone (LH) and follicle stimulating hormone (FSH). Testosterone is converted to the more active form, dihydrotestosterone, (DHT), within the prostate by 5α-reductase. Adrenal androgens also contribute about 20% of total DHT in the rat prostate, compared to 40% of that in 65-year-old men (Labrie et. al. Clin. Invest. Med., 45, 475492 (1993)). However, this is not a major pathway, since in both animals and humans, castration leads to almost complete involution of the prostate and seminal vesicles without concomitant adrenalectomy. Therefore, under normal conditions, the adrenals do not support significant growth of prostate tissues (Luke, M. C. and Coffey, D. S. “The Physiology of Reproduction” ed. By E. Knobil and J. D. Neill, 1, 1435-1487 (1994)). Since the male sex organs and the levator ani are the tissues most responsive to modulation of the androgen activity, this model is used to determine the activity of compounds that modulate the androgen receptor pathway in mature rats.

Along with its mitogenic activity on tissues such as prostate, seminal vesicle and muscle, testosterone also serves as a negative regulator for its own biosynthesis. Testosterone production in the Leydig cells of the testis is controlled by the level of circulating LH released from the pituitary gland. LH levels are themselves controlled by the level of LHR.H produced in the hypothalmic region. Testosterone levels in the blood serve to inhibit the secretion of LHRH and subsequently reduce levels of LH and ultimately the levels of circulating testosterone levels. By measuring blood levels of LH as they are effected by test compounds, it is possible to determine the level of agonist or antagonist activity of said compounds at the hypothalamic axis of this endocrine cycle.

Matched sets of Harlan Sprague-Dawley rats (40-42 days old, 180-220 g), were dosed orally by gavage (p.o.) with the test compounds in dissolved/suspensions of 80% PEG 400 and 20% Tween 20 (PEGTW] for 14 days. Two control groups, one intact and one castrated were dosed orally only with the PEGTW vehicle. Animals were dosed (v/w) at 0.5 ml of vehicle /100 g body weight. Experimental groups were as follows:

-   -   1. Intact vehicle (p.o., PEGTW, QD)     -   2. Control vehicle (p.o., PEGTW, QD)     -   3. Biacalutamide (Casodex, a recognized antiandrogen, as a         reference compound) or a test compound, p.o. in PEGTW QD. (in a         range of doses).

At the end of the 14-day treatment, the animals were sacrificed, and the ventral prostate, the seminal vesicles, and the levator ani were removed surgically and weighed. To compare data from different experiments, the organs weights were first standardized as mg per 100 g of body weight, and expressed as a percentage of the value of the respective organ in the intact group.

Rat luteinizing hormone (rLH) is quantitatively determined with the Biotrak [¹²⁵I] kit (Amersham Pharmacia Biotek), following the manufacturer directions. The assay is based on the competition by the LH present in the serum of the binding of [¹²⁵I] rLH to an Amerlex-M bead/antibody suspension. The radioactivity that remains after incubation with the serum and subsequent washes is extrapolated into a standard curve to obtain a reading in ng/ml.

The gain and loss of sexual organ and levator ani weight reflect the changes of the cell number (DNA content) and cell mass (protein content), depending upon the serum androgen concentration (Okuda et al., J. Urol., 145, 188-191 (1991)). Therefore, measurement of organ wet weight is sufficient to indicate the bioactivity of androgens and androgen antagonist. In the mature rats assay, active agonist agents will have no effect or will increase the weight of one or more of the androgen responsive organs (levator ani, prostate, seminal vesicle) and will have no effect or a suppressive effect on LH secretion. Compounds with antagonist activity will decrease the weight of one or more of the androgen responsive organs (levator ani, prostate, seminal vesicle) and will have no effect or a reduced suppressive effect on LH secretion.

Example 10 MDA PCa2b Human Prostate Zenograft Assay

For in vivo antitumor testing, MDA-PCa-2b human prostate tumors were maintained in Balb/c nu/nu nude mice. Tumors were propagated as subcutaneous transplants in adult male nude mice (4-6 weeks old) using tumor fragments obtained from donor mice. Tumor passage occurred every 5-6 weeks.

For antitumor efficacy trial, the required number of animals needed to detect a meaningful response were pooled at the start of the experiment and each was given a subcutaneous implant of a tumor fragment (˜50 mg) with a 13-gauge trocar. Tumors were allowed to grow to approximately 100-200 mg (tumors outside the range were excluded) and animals were evenly distributed to various treatment and control groups. Treatment of each animal was based on individual body weight. Treated animals were checked daily for treatment related toxicity/mortality. Each group of animals was weighed before the initiation of treatment (Wt1) and then again following the last treatment dose (Wt2). The difference in body weight (Wt2-Wt1) provides a measure of treatment-related toxicity.

Tumor response was determined by measurement of tumors with a caliper twice a week, until the tumors reach a predetermined “target” size of 0.5 gm. Tumor weights (mg) were estimated from the formula: Tumor weight=(length×width2)÷2

Tumor response end-point was expressed in terms of tumor growth inhibition (% T/C), defined as the ratio of median tumor weights of the treated tumors (T) to that of the control group (C).

To estimate tumor cell kill, the tumor volume doubling time was first calculated with the formula: TVDT=Median time (days) for control tumors to reach target size−Median time (days) for control tumors to reach half the target size s and, Log cell kill was then calculated with the formula Log cell kill=(T−C)÷(3.32×TVDT)

Statistical evaluations of data were performed using Gehan's generalized Wilcoxon test.

Example 11 CWR22 Human Prostate Zenograft Assay

In vivo antitumor testing was also performed with CWR22 human prostate tumors maintained in Balb/c nu/nu nude mice. Tumors were propagated as subcutaneous transplants in adult male nude mice (4-6 weeks old) using tumor fragments obtained from donor mice. Tumor passage occurred every 5-6 weeks.

For antitumor efficacy trial, the required number of animals needed to detect a meaningful response were pooled at the start of the experiment and each was given a subcutaneous implant of a tumor fragment (˜50 mg) with a 1 3-gauge trocar. Tumors were allowed to grow to approximately 100-200 mg (tumors outside the range were excluded) and animals were evenly distributed to various treatment and control groups. Treatment of each animal was based on individual body weight. Treated animals were checked daily for treatment related toxicity/mortality. Each group of animals was weighed before the initiation of treatment (Wt1) and then again following the last treatment dose (Wt2). The difference in body weight (Wt2-Wt1) provides a measure of treatment-related toxicity.

Tumor response was determined by measurement of tumors with a caliper twice a week, until the tumors reach a predetermined “target” size of 0.5 gm. Tumor weights (mg) were estimated from the formula: Tumor weight=(length×width2)÷2

Tumor response end-point was expressed in terms of tumor growth inhibition (% T/C), defined as the ratio of median tumor weights of the treated tumors (T) to that of the control group (C).

To estimate tumor cell kill, the tumor volume doubling time was first calculated with the formula: TVDT=Median time (days) for control tumors to reach target size−Median time (days) for control tumors to reach half the target size s Log cell kill was calculated with the formula: Log cell kill=(T−C)÷(3.32×TVDT)

Statistical evaluations of data were performed using Gehan's generalized Wilcoxon test.

Example 12 Dunning R3327H Rat Prostate Tumor Assay

Dunning R3327H prostate tumor is a spontaneously derived, well 15 differentiated androgen responsive adenocarcinoma of the prostate (Smolev et al. Cancer Treat Rep. 61, 273-287 (1977)). The growth of the R3327H subline has been selected for its highly androgen-dependent and reproducible growth in intact male rats. Therefore, this model and other sublines of this tumor have been widely used to evaluate in vivo antitumor activities of antiandrogens such as flutamide and bacilutamide/Casodex (Maucher A., and von Angerer, J. Cancer Res. Clin,. Oncol. 119, 669-674 (1993), Furr B. J. A. Euro. URL. 18(suppl. 3), 2-9 (1990), Shain S. A. and Huot R I. J. Steriod Biochem. 31, 711-718 (1988)). For this assay, Dunning tumor pieces (about 4×4 mm) are transplanted subcutaneously to the flank of mature male Copenhagen rats (6-7 weeks old, Harlan-Sprague Dawley, Indianapolis, Md.). About 6 weeks after the implantation, the animals with tumors of measurable size (about 80-120 mm²) are randomized into treatment groups (8-10 rats/group) and the treatments are initiated. One group of the rats are castrated to serve as the negative control of tumor growth. Animals are treated daily with test compounds, standard antiandrogens such as bacilutamide or vehicle (control) for an average of 10 to 14 weeks. Test compounds are dissolved in a vehicle of (2.5 ml/kg of body weight) 10% polyethylene glycol and 0.05% Tween-80 in 1% carboxymethyl cellulose, PEG/CMC, (Sigma, St Louis, Mo.). Typical experiments include three groups of three escalating doses for each standard or test compound (in a range of 300-3 mg/kg).

Tumors in the vehicle group the control group tumors reach a size of 1500 to 2500 mM³. In contrast, the castrated animal group typically shows tumor stasis over the 14 weeks of observation. Animals treated orally with 20 mg/kg of bicalutamide or flutamide are expected to show 40% reduction in tumor volumes compared to controls after 14 weeks of treatment. The size of tumors are measured weekly by vernier caliper (Froboz, Switzerland), taking perpendicular measurements of length and width. Tumor volumes are measured in mm³ using the formula: Length×Width×Height=Volume. Statistical differences between treatment groups and control are evaluated using multiple ANOVA analysis followed by one tail non-parametric Student t test.

Example 13 Dunning R3327H Rat Prostate Tumor and Wet Prostate Weight Assay

Dunning tumor pieces (about 4×4 mm), as described in Example 11, are transplanted subcutaneously to the flank of mature male Copenhagen rats (6-7 weeks old, Harlan-Sprague Dawley, Indianapolis, Md.). About 6 weeks after the implantation, the animals with tumors of measurable size (about 80-120 mm²) are randomized into treatment groups (8-10 rats/group) and the treatments are initiated. One group of the rats are castrated to serve as the negative control of tumor growth. Animals are treated daily with test compounds, standard antiandrogens such as bacilutamide or vehicle (control) for an average of 10 to 14 weeks. Test compounds are dissolved in a vehicle of (2.5 ml/kg of body weight) 10% polyethylene glycol and 0.05% Tween-80 in 1% carboxymethyl cellulose, PEG/CMC, (Sigma, St Louis, Mo.). Typical therapeutic experiments would include three groups of three escalating doses for each standard or test compound (in a range of 300-3 mg/kg).

Tumors in the vehicle group the control group tumors reach a size of 1500 to 2500 mm³. In contrast, the castrated animal group typically shows tumor stasis over the 14 weeks of observation. Animals treated orally with 20 mg/kg of bicalutamide or flutamide are expected to show 40% reduction in tumor volumes compared to control after 14 weeks of treatment. The size of tumors are measured weekly by vernier caliper (Froboz, Switzerland), taking perpendicular measurements of length and width. Tumor volumes are measured in mm³ using the formula: Length×Width×Height=Volume. Statistical differences between treatment groups and control are evaluated using multiple ANOVA analysis followed by one tail non-parametric Student t test.

At the end of the treatment, the animals were sacrificed, and the ventral prostate, the seminal vesicles, and the levator ani were removed surgically and weighed. To compare data from different experiments, the organs weights were first standardized as mg per 100 g of body weight, and expressed as a percentage of the value of the respective organ in the intact group.

Rat luteinizing hormone (rLH) can be quantitatively determined in these animals with the Biotrak [¹²⁵I] kit (Amersham Pharmacia Biotek), following the manufacturer directions. The assay is based on the competition by the LH present in the serum of the binding of [¹²⁵ I] rLH to an Amerlex-M bead/antibody suspension. The radioactivity that remains after incubation with the serum and subsequent washes is extrapolated into a standard curve to obtain a reading in ng/ml.

The gain and loss of sexual organ and levator ani weight reflect the changes of the cell number (DNA content) and cell mass (protein content), depending upon the serum androgen concentration (Okuda et al., J. Urol., 145, 188-191 (1991)). Therefore, measurement of organ wet weight is sufficient to indicate the bioactivity of androgens and androgen antagonist. In this rat assay, active agonist agents will have no effect or will increase the weight of one or more of the androgen responsive organs (levator ani, prostate, seminal vesicle) and will have no effect or a suppressive effect on LH secretion. Compounds with antagonist activity will decrease the weight of one or more of the androgen responsive organs (levator ani, prostate, seminal vesicle) and will have no effect or a reduced suppressive effect on LH secretion. TABLE A ATOM 1 CB ILE 672 15.585 25.993 23.410 1.00 31.24 ATOM 2 CG2 ILE 672 14.852 27.128 24.154 1.00 30.88 ATOM 3 CG1 ILE 672 16.008 24.898 24.385 1.00 31.15 ATOM 4 CD1 ILE 672 16.804 25.398 25.547 1.00 31.82 ATOM 5 C ILE 672 15.338 24.127 21.758 1.00 30.41 ATOM 6 O ILE 672 16.366 24.192 21.075 1.00 30.37 ATOM 7 N ILE 672 13.327 25.032 22.857 1.00 31.02 ATOM 8 CA ILE 672 14.670 25.387 22.310 1.00 30.84 ATOM 9 N PHE 673 14.730 22.987 22.058 1.00 29.49 ATOM 10 CA PHE 673 15.233 21.694 21.628 1.00 28.89 ATOM 11 CB PHE 673 14.309 20.587 22.143 1.00 28.59 ATOM 12 CG PHE 673 14.827 19.211 21.890 1.00 28.37 ATOM 13 CD1 PHE 673 15.903 18.721 22.616 1.00 28.33 ATOM 14 CD2 PHE 673 14.259 18.412 20.900 1.00 27.95 ATOM 15 CE1 PHE 673 16.411 17.447 22.358 1.00 28.35 ATOM 16 CE2 PHE 673 14.753 17.151 20.634 1.00 27.76 ATOM 17 CZ PHE 673 15.831 16.665 21.361 1.00 28.28 ATOM 18 C PHE 673 15.368 21.591 20.108 1.00 28.44 ATOM 19 O PHE 673 16.387 21.137 19.594 1.00 28.30 ATOM 20 N LEU 674 14.334 22.000 19.387 1.00 28.13 ATOM 21 CA LEU 674 14.393 21.950 17.940 1.00 27.74 ATOM 22 CB LEU 674 13.033 22.315 17.337 1.00 28.50 ATOM 23 CG LEU 674 12.094 21.110 17.212 1.00 29.52 ATOM 24 CD1 LEU 674 12.732 20.084 16.273 1.00 29.84 ATOM 25 CD2 LEU 674 11.846 20.487 18.590 1.00 29.46 ATOM 26 C LEU 674 15.472 22.876 17.402 1.00 27.02 ATOM 27 O LEU 674 16.174 22.529 16.452 1.00 26.73 ATOM 28 N ASN 675 15.605 24.048 18.017 1.00 26.63 ATOM 29 CA ASN 675 16.595 25.031 17.592 1.00 26.23 ATOM 30 CB ASN 675 16.584 26.238 18.547 1.00 26.53 ATOM 31 CG ASN 675 15.229 26.955 18.575 1.00 26.69 ATOM 32 OD1 ASN 675 14.771 27.475 17.568 1.00 25.27 ATOM 33 ND2 ASN 675 14.587 26.973 19.740 1.00 28.17 ATOM 34 C ASN 675 17.971 24.371 17.578 1.00 25.92 ATOM 35 O ASN 675 18.716 24.475 16.599 1.00 25.60 ATOM 36 N VAL 676 18.284 23.666 18.663 1.00 25.22 ATOM 37 CA VAL 676 19.561 22.979 18.800 1.00 24.86 ATOM 38 CB VAL 676 19.667 22.306 20.180 1.00 24.40 ATOM 39 CG1 VAL 676 20.987 21.567 20.300 1.00 23.97 ATOM 40 CG2 VAL 676 19.525 23.354 21.271 1.00 24.34 ATOM 41 C VAL 676 19.777 21.920 17.715 1.00 24.78 ATOM 42 O VAL 676 20.857 21.835 17.123 1.00 23.74 ATOM 43 N LEU 677 18.742 21.115 17.471 1.00 24.81 ATOM 44 CA LEU 677 18.804 20.055 16.471 1.00 25.01 ATOM 45 CB LEU 677 17.540 19.181 16.561 1.00 24.69 ATOM 46 CG LEU 677 17.645 17.937 17.468 1.00 25.09 ATOM 47 CD1 LEU 677 18.155 18.298 18.847 1.00 24.53 ATOM 48 CD2 LEU 677 16.287 17.270 17.570 1.00 24.88 ATOM 49 C LEU 677 19.018 20.560 15.037 1.00 24.85 ATOM 50 O LEU 677 19.770 19.967 14.274 1.00 24.74 ATOM 51 N GLU 678 18.362 21.655 14.675 1.00 25.51 ATOM 52 CA GLU 678 18.504 22.232 13.334 1.00 25.67 ATOM 53 CB GLU 678 17.413 23.284 13.103 1.00 27.59 ATOM 54 CG GLU 678 17.732 24.340 12.046 1.00 30.04 ATOM 55 CD GLU 678 16.621 25.379 11.918 1.00 32.44 ATOM 56 OE1 GLU 678 16.830 26.423 11.237 1.00 33.09 ATOM 57 OE2 GLU 678 15.534 25.140 12.505 1.00 33.23 ATOM 58 C GLU 678 19.874 22.873 13.187 1.00 24.83 ATOM 59 O GLU 678 20.541 22.715 12.171 1.00 24.31 ATOM 60 N ALA 679 20.286 23.591 14.224 1.00 24.77 ATOM 61 CA ALA 679 21.571 24.273 14.244 1.00 24.70 ATOM 62 CB ALA 679 21.703 25.072 15.515 1.00 24.21 ATOM 63 C ALA 679 22.744 23.315 14.125 1.00 25.17 ATOM 64 O ALA 679 23.752 23.637 13.499 1.00 25.65 ATOM 65 N ILE 680 22.623 22.137 14.722 1.00 25.38 ATOM 66 CA ILE 680 23.716 21.172 14.676 1.00 25.89 ATOM 67 CB ILE 680 23.829 20.417 15.999 1.00 25.44 ATOM 68 CG2 ILE 680 23.886 21.427 17.143 1.00 24.98 ATOM 69 CG1 ILE 680 22.649 19.439 16.135 1.00 24.49 ATOM 70 CD1 ILE 680 22.583 18.702 17.442 1.00 24.31 ATOM 71 C ILE 680 23.620 20.140 13.563 1.00 26.38 ATOM 72 O ILE 680 24.482 19.270 13.472 1.00 26.49 ATOM 73 N GLU 681 22.586 20.227 12.728 1.00 26.84 ATOM 74 CA GLU 681 22.414 19.262 11.641 1.00 28.06 ATOM 75 CB GLU 681 21.074 19.473 10.946 1.00 28.71 ATOM 76 CG GLU 681 20.790 18.472 9.850 1.00 29.82 ATOM 77 CD GLU 681 20.527 17.083 10.393 1.00 31.25 ATOM 78 OE1 GLU 681 20.168 16.187 9.588 1.00 32.01 ATOM 79 OE2 GLU 681 20.677 16.887 11.623 1.00 31.32 ATOM 80 C GLU 681 23.533 19.348 10.605 1.00 28.62 ATOM 81 O GLU 681 23.755 20.398 9.993 1.00 29.09 ATOM 82 N PRO 682 24.247 18.235 10.384 1.00 28.78 ATOM 83 CD PRO 682 24.071 16.919 11.017 1.00 28.67 ATOM 84 CA PRO 682 25.348 18.198 9.420 1.00 28.68 ATOM 85 CB PRO 682 25.864 16.764 9.533 1.00 28.60 ATOM 86 CG PRO 682 25.440 16.337 10.882 1.00 28.67 ATOM 87 C PRO 682 24.886 18.505 8.004 1.00 28.93 ATOM 88 O PRO 682 23.765 18.165 7.620 1.00 29.17 ATOM 89 N GLY 683 25.760 19.141 7.233 1.00 28.83 ATOM 90 CA GLY 683 25.438 19.454 5.855 1.00 28.82 ATOM 91 C GLY 683 25.843 18.296 4.951 1.00 29.01 ATOM 92 O GLY 683 26.077 17.187 5.421 1.00 28.29 ATOM 93 N VAL 684 25.935 18.569 3.652 1.00 29.33 ATOM 94 CA VAL 684 26.293 17.569 2.648 1.00 29.92 ATOM 95 CB VAL 684 26.276 18.182 1.223 1.00 30.47 ATOM 96 CG1 VAL 684 26.393 17.069 0.166 1.00 30.24 ATOM 97 CG2 VAL 684 25.004 19.006 1.025 1.00 29.99 ATOM 98 C VAL 684 27.666 16.933 2.855 1.00 29.77 ATOM 99 O VAL 684 28.602 17.578 3.308 1.00 29.87 ATOM 100 N VAL 685 27.768 15.657 2.512 1.00 29.69 ATOM 101 CA VAL 685 29.014 14.922 2.631 1.00 29.84 ATOM 102 CB VAL 685 28.973 13.936 3.834 1.00 29.89 ATOM 103 CG1 VAL 685 30.357 13.368 4.084 1.00 29.18 ATOM 104 CG2 VAL 685 28.441 14.642 5.088 1.00 29.80 ATOM 105 C VAL 685 29.207 14.130 1.333 1.00 30.42 ATOM 106 O VAL 685 28.378 13.291 0.975 1.00 30.40 ATOM 107 N CYS 686 30.287 14.408 0.615 1.00 30.75 ATOM 108 CA CYS 686 30.551 13.698 −0.624 1.00 31.29 ATOM 109 CB CYS 686 31.219 14.636 −1.630 1.00 31.47 ATOM 110 SG CYS 686 30.172 16.063 −2.105 1.00 33.49 ATOM 111 C CYS 686 31.415 12.459 −0.366 1.00 31.56 ATOM 112 O CYS 686 32.266 12.458 0.523 1.00 31.13 ATOM 113 N ALA 687 31.180 11.405 −1.147 1.00 31.88 ATOM 114 CA ALA 687 31.905 10.157 −0.987 1.00 32.66 ATOM 115 CB ALA 687 31.072 8.989 −1.531 1.00 32.89 ATOM 116 C ALA 687 33.275 10.160 −1.636 1.00 33.22 ATOM 117 O ALA 687 34.138 9.365 −1.259 1.00 33.23 ATOM 118 N GLY 688 33.476 11.051 −2.602 1.00 33.83 ATOM 119 CA GLY 688 34.752 11.123 −3.287 1.00 34.36 ATOM 120 C GLY 688 34.838 10.039 −4.345 1.00 35.22 ATOM 121 O GLY 688 35.925 9.648 −4.776 1.00 35.29 ATOM 122 N HIS 689 33.681 9.540 −4.762 1.00 35.46 ATOM 123 CA HIS 689 33.639 8.497 −5.773 1.00 36.39 ATOM 124 CB HIS 689 32.328 7.713 −5.650 1.00 36.01 ATOM 125 CG HIS 689 32.136 6.683 −6.716 1.00 35.72 ATOM 126 CD2 HIS 689 32.433 5.361 −6.742 1.00 35.47 ATOM 127 ND1 HIS 689 31.590 6.977 −7.946 1.00 35.68 ATOM 128 CE1 HIS 689 31.557 5.882 −8.684 1.00 35.61 ATOM 129 NE2 HIS 689 32.063 4.887 −7.976 1.00 35.45 ATOM 130 C HIS 689 33.776 9.088 −7.176 1.00 37.25 ATOM 131 O HIS 689 33.125 10.078 −7.512 1.00 36.54 ATOM 132 N ASP 690 34.643 8.492 −7.988 1.00 38.49 ATOM 133 CA ASP 690 34.832 8.967 −9.353 1.00 40.16 ATOM 134 CB ASP 690 36.285 8.755 −9.803 1.00 40.96 ATOM 135 CG ASP 690 36.766 7.342 −9.580 1.00 42.31 ATOM 136 OD1 ASP 690 37.918 7.030 −9.977 1.00 42.84 ATOM 137 OD2 ASP 690 35.994 6.541 −9.004 1.00 43.23 ATOM 138 C ASP 690 33.870 8.242 −10.293 1.00 40.81 ATOM 139 O ASP 690 33.928 7.020 −10.434 1.00 41.00 ATOM 140 N ASN 691 32.972 8.997 −10.919 1.00 41.55 ATOM 141 CA ASN 691 31.992 8.419 −11.838 1.00 42.66 ATOM 142 CB ASN 691 30.917 9.450 −12.195 1.00 42.57 ATOM 143 CG ASN 691 30.002 9.782 −11.031 1.00 42.88 ATOM 144 OD1 ASN 691 29.206 10.721 −11.115 1.00 43.45 ATOM 145 ND2 ASN 691 30.096 9.014 −9.946 1.00 42.16 ATOM 146 C ASN 691 32.620 7.894 −13.132 1.00 43.34 ATOM 147 O ASN 691 31.931 7.304 −13.962 1.00 43.67 ATOM 148 N ASN 692 33.918 8.120 −13.307 1.00 43.99 ATOM 149 CA ASN 692 34.626 7.661 −14.499 1.00 44.50 ATOM 150 CB ASN 692 36.045 8.233 −14.521 1.00 44.96 ATOM 151 CG ASN 692 36.163 9.552 −13.757 1.00 45.75 ATOM 152 OD1 ASN 692 35.869 9.625 −12.558 1.00 45.86 ATOM 153 ND2 ASN 692 36.604 10.598 −14.449 1.00 45.86 ATOM 154 C ASN 692 34.703 6.138 −14.431 1.00 44.51 ATOM 155 O ASN 692 35.016 5.467 −15.415 1.00 44.65 ATOM 156 N GLN 693 34.401 5.620 −13.247 1.00 44.30 ATOM 157 CA GLN 693 34.432 4.195 −12.933 1.00 44.20 ATOM 158 CB GLN 693 34.091 4.004 −11.452 1.00 44.34 ATOM 159 CG GLN 693 34.453 2.664 −10.864 1.00 44.63 ATOM 160 CD GLN 693 35.935 2.552 −10.548 1.00 44.93 ATOM 161 OE1 GLN 693 36.544 3.501 −10.055 1.00 44.97 ATOM 162 NE2 GLN 693 36.518 1.383 −10.813 1.00 44.61 ATOM 163 C GLN 693 33.469 3.362 −13.765 1.00 43.84 ATOM 164 O GLN 693 32.271 3.634 −13.802 1.00 44.50 ATOM 165 N PRO 694 33.976 2.324 −14.439 1.00 43.36 ATOM 166 CD PRO 694 35.372 1.893 −14.620 1.00 43.36 ATOM 167 CA PRO 694 33.069 1.503 −15.238 1.00 43.02 ATOM 168 CB PRO 694 34.021 0.607 −16.027 1.00 43.07 ATOM 169 CG PRO 694 35.200 0.484 −15.122 1.00 43.30 ATOM 170 C PRO 694 32.111 0.717 −14.338 1.00 42.75 ATOM 171 O PRO 694 31.333 −0.121 −14.816 1.00 43.47 ATOM 172 N ASP 695 32.174 0.993 −13.036 1.00 41.47 ATOM 173 CA ASP 695 31.309 0.342 −12.056 1.00 40.30 ATOM 174 CB ASP 695 29.858 0.406 −12.521 1.00 40.36 ATOM 175 CG ASP 695 28.998 1.238 −11.610 1.00 40.64 ATOM 176 OD1 ASP 695 27.988 1.793 −12.085 1.00 40.36 ATOM 177 OD2 ASP 695 29.329 1.329 −10.411 1.00 41.36 ATOM 178 C ASP 695 31.701 −1.103 −11.772 1.00 39.59 ATOM 179 O ASP 695 32.158 −1.829 −12.661 1.00 39.66 ATOM 180 N SER 696 31.519 −1.511 −10.523 1.00 37.97 ATOM 181 CA SER 696 31.870 −2.854 −10.093 1.00 36.83 ATOM 182 CB SER 696 33.334 −3.141 −10.409 1.00 36.85 ATOM 183 OG SER 696 34.170 −2.172 −9.803 1.00 36.78 ATOM 184 C SER 696 31.658 −2.985 −8.595 1.00 35.83 ATOM 185 O SER 696 31.175 −2.063 −7.938 1.00 35.84 ATOM 186 N PHE 697 32.034 −4.135 −8.058 1.00 34.43 ATOM 187 CA PHE 697 31.886 −4.376 −6.639 1.00 33.31 ATOM 188 CB PHE 697 31.970 −5.872 −6.353 1.00 32.81 ATOM 189 CG PHE 697 31.755 −6.225 −4.910 1.00 32.02 ATOM 190 CD1 PHE 697 30.601 −5.839 −4.253 1.00 31.59 ATOM 191 CD2 PHE 697 32.712 −6.939 −4.207 1.00 31.69 ATOM 192 CE1 PHE 697 30.411 −6.162 −2.913 1.00 31.45 ATOM 193 CE2 PHE 697 32.522 −7.264 −2.871 1.00 31.30 ATOM 194 CZ PHE 697 31.375 −6.875 −2.227 1.00 30.74 ATOM 195 C PHE 697 32.959 −3.634 −5.849 1.00 32.77 ATOM 196 O PHE 697 32.663 −2.997 −4.846 1.00 32.24 ATOM 197 N ALA 698 34.199 −3.701 −6.326 1.00 32.43 ATOM 198 CA ALA 698 35.333 −3.059 −5.659 1.00 32.06 ATOM 199 CB ALA 698 36.628 −3.462 −6.351 1.00 32.14 ATOM 200 C ALA 698 35.252 −1.533 −5.562 1.00 31.78 ATOM 201 O ALA 698 35.503 −0.958 −4.506 1.00 31.59 ATOM 202 N ALA 699 34.903 −0.883 −6.663 1.00 31.20 ATOM 203 CA ALA 699 34.799 0.569 −6.692 1.00 30.50 ATOM 204 CB ALA 699 34.627 1.035 −8.125 1.00 30.71 ATOM 205 C ALA 699 33.658 1.117 −5.837 1.00 30.14 ATOM 206 O ALA 699 33.793 2.161 −5.192 1.00 29.89 ATOM 207 N LEU 700 32.525 0.425 −5.856 1.00 29.56 ATOM 208 CA LEU 700 31.353 0.846 −5.092 1.00 29.08 ATOM 209 CB LEU 700 30.166 −0.054 −5.431 1.00 29.19 ATOM 210 CG LEU 700 29.075 0.521 −6.332 1.00 30.19 ATOM 211 CD1 LEU 700 29.649 1.531 −7.321 1.00 29.81 ATOM 212 CD2 LEU 700 28.377 −0.636 −7.053 1.00 30.55 ATOM 213 C LEU 700 31.600 0.817 −3.587 1.00 28.70 ATOM 214 O LEU 700 31.687 1.858 −2.939 1.00 28.63 ATOM 215 N LEU 701 31.718 −0.387 −3.045 1.00 28.11 ATOM 216 CA LEU 701 31.944 −0.580 −1.624 1.00 28.02 ATOM 217 CB LEU 701 32.126 −2.068 −1.325 1.00 26.80 ATOM 218 CG LEU 701 30.785 −2.788 −1.243 1.00 26.52 ATOM 219 CD1 LEU 701 29.977 −2.199 −0.085 1.00 25.48 ATOM 220 CD2 LEU 701 30.028 −2.659 −2.562 1.00 25.47 ATOM 221 C LEU 701 33.126 0.200 −1.079 1.00 28.03 ATOM 222 O LEU 701 33.065 0.744 0.023 1.00 27.83 ATOM 223 N SER 702 34.200 0.250 −1.857 1.00 28.39 ATOM 224 CA SER 702 35.397 0.965 −1.446 1.00 28.48 ATOM 225 CB SER 702 36.508 0.793 −2.478 1.00 28.93 ATOM 226 OG SER 702 37.745 1.184 −1.922 1.00 30.72 ATOM 227 C SER 702 35.078 2.440 −1.266 1.00 27.81 ATOM 228 O SER 702 35.585 3.072 −0.345 1.00 28.06 ATOM 229 N SER 703 34.233 2.984 −2.140 1.00 27.29 ATOM 230 CA SER 703 33.850 4.388 −2.040 1.00 26.12 ATOM 231 CB SER 703 33.349 4.916 −3.385 1.00 26.33 ATOM 232 OG SER 703 31.941 5.056 −3.376 1.00 28.04 ATOM 233 C SER 703 32.762 4.510 −0.981 1.00 25.38 ATOM 234 O SER 703 32.637 5.543 −0.333 1.00 25.43 ATOM 235 N LEU 704 31.975 3.453 −0.791 1.00 24.62 ATOM 236 CA LEU 704 30.945 3.492 0.247 1.00 23.83 ATOM 237 CB LEU 704 29.987 2.306 0.122 1.00 23.34 ATOM 238 CG LEU 704 28.843 2.445 −0.888 1.00 22.81 ATOM 239 CD1 LEU 704 27.962 1.199 −0.844 1.00 22.05 ATOM 240 CD2 LEU 704 28.010 3.673 −0.530 1.00 22.63 ATOM 241 C LEU 704 31.629 3.473 1.614 1.00 23.67 ATOM 242 O LEU 704 31.212 4.171 2.537 1.00 23.27 ATOM 243 N ASN 705 32.681 2.663 1.728 1.00 23.68 ATOM 244 CA ASN 705 33.454 2.556 2.959 1.00 23.54 ATOM 245 CB ASN 705 34.597 1.546 2.808 1.00 22.57 ATOM 246 CG ASN 705 34.135 0.107 2.920 1.00 22.79 ATOM 247 OD1 ASN 705 34.865 −0.826 2.553 1.00 22.63 ATOM 248 ND2 ASN 705 32.932 −0.089 3.447 1.00 21.75 ATOM 249 C ASN 705 34.051 3.920 3.248 1.00 23.95 ATOM 250 O ASN 705 34.095 4.355 4.395 1.00 24.17 ATOM 251 N GLU 706 34.513 4.599 2.204 1.00 24.44 ATOM 252 CA GLU 706 35.110 5.907 2.402 1.00 25.29 ATOM 253 CB GLU 706 35.729 6.452 1.118 1.00 26.16 ATOM 254 CG GLU 706 36.404 7.788 1.340 1.00 27.89 ATOM 255 CD GLU 706 37.478 7.726 2.428 1.00 29.25 ATOM 256 OE1 GLU 706 37.936 8.799 2.881 1.00 29.77 ATOM 257 OE2 GLU 706 37.868 6.604 2.831 1.00 30.55 ATOM 258 C GLU 706 34.063 6.874 2.884 1.00 25.01 ATOM 259 O GLU 706 34.352 7.747 3.694 1.00 25.76 ATOM 260 N LEU 707 32.842 6.714 2.385 1.00 24.51 ATOM 261 CA LEU 707 31.749 7.585 2.778 1.00 24.01 ATOM 262 CB LEU 707 30.511 7.333 1.912 1.00 23.04 ATOM 263 CG LEU 707 29.345 8.281 2.195 1.00 22.39 ATOM 264 CD1 LEU 707 29.746 9.740 1.906 1.00 21.36 ATOM 265 CD2 LEU 707 28.168 7.883 1.343 1.00 22.62 ATOM 266 C LEU 707 31.382 7.379 4.230 1.00 24.28 ATOM 267 O LEU 707 31.075 8.333 4.941 1.00 24.68 ATOM 268 N GLY 708 31.414 6.126 4.666 1.00 24.48 ATOM 269 CA GLY 708 31.052 5.818 6.033 1.00 24.27 ATOM 270 C GLY 708 31.987 6.483 7.013 1.00 24.28 ATOM 271 O GLY 708 31.557 6.976 8.043 1.00 24.53 ATOM 272 N GLU 709 33.271 6.488 6.688 1.00 24.50 ATOM 273 CA GLU 709 34.278 7.094 7.548 1.00 25.04 ATOM 274 CB GLU 709 35.676 6.727 7.037 1.00 26.02 ATOM 275 CG GLU 709 36.846 7.291 7.830 1.00 28.27 ATOM 276 CD GLU 709 36.963 6.712 9.238 1.00 30.06 ATOM 277 OE1 GLU 709 36.142 7.085 10.116 1.00 30.97 ATOM 278 OE2 GLU 709 37.882 5.884 9.466 1.00 30.27 ATOM 279 C GLU 709 34.083 8.613 7.571 1.00 24.49 ATOM 280 O GLU 709 34.176 9.240 8.624 1.00 24.26 ATOM 281 N ARG 710 33.792 9.198 6.414 1.00 23.72 ATOM 282 CA ARG 710 33.581 10.639 6.342 1.00 23.54 ATOM 283 CB ARG 710 33.426 11.097 4.885 1.00 23.22 ATOM 284 CG ARG 710 34.715 10.999 4.073 1.00 23.82 ATOM 285 CD ARG 710 34.524 11.398 2.626 1.00 24.53 ATOM 286 NE ARG 710 35.807 11.550 1.952 1.00 25.26 ATOM 287 CZ ARG 710 35.967 12.064 0.737 1.00 25.57 ATOM 288 NH1 ARG 710 34.920 12.481 0.036 1.00 25.76 ATOM 289 NH2 ARG 710 37.183 12.180 0.226 1.00 26.08 ATOM 290 C ARG 710 32.365 11.065 7.152 1.00 23.53 ATOM 291 O ARG 710 32.404 12.074 7.867 1.00 23.75 ATOM 292 N GLN 711 31.286 10.294 7.052 1.00 23.34 ATOM 293 CA GLN 711 30.081 10.627 7.786 1.00 23.12 ATOM 294 CB GLN 711 28.893 9.819 7.274 1.00 22.95 ATOM 295 CG GLN 711 28.250 10.414 6.037 1.00 22.79 ATOM 296 CD GLN 711 26.952 9.724 5.655 1.00 23.04 ATOM 297 OE1 GLN 711 26.165 9.329 6.519 1.00 23.05 ATOM 298 NE2 GLN 711 26.715 9.587 4.356 1.00 22.64 ATOM 299 C GLN 711 30.285 10.400 9.273 1.00 23.07 ATOM 300 O GLN 711 29.672 11.072 10.092 1.00 22.74 ATOM 301 N LEU 712 31.159 9.458 9.617 1.00 23.12 ATOM 302 CA LEU 712 31.459 9.179 11.018 1.00 22.87 ATOM 303 CB LEU 712 32.475 8.046 11.131 1.00 22.35 ATOM 304 CG LEU 712 33.072 7.933 12.536 1.00 22.63 ATOM 305 CD1 LEU 712 31.940 7.890 13.580 1.00 22.01 ATOM 306 CD2 LEU 712 33.969 6.701 12.615 1.00 22.12 ATOM 307 C LEU 712 32.029 10.452 11.651 1.00 23.01 ATOM 308 O LEU 712 31.681 10.822 12.784 1.00 22.93 ATOM 309 N VAL 713 32.907 11.119 10.907 1.00 22.87 ATOM 310 CA VAL 713 33.498 12.362 11.374 1.00 22.95 ATOM 311 CB VAL 713 34.344 13.042 10.255 1.00 23.84 ATOM 312 CG1 VAL 713 34.691 14.487 10.648 1.00 22.62 ATOM 313 CG2 VAL 713 35.613 12.226 9.988 1.00 23.07 ATOM 314 C VAL 713 32.384 13.319 11.796 1.00 22.67 ATOM 315 O VAL 713 32.475 13.957 12.847 1.00 22.99 ATOM 316 N HIS 714 31.333 13.404 10.981 1.00 22.28 ATOM 317 CA HIS 714 30.211 14.302 11.266 1.00 22.20 ATOM 318 CB HIS 714 29.444 14.647 9.980 1.00 22.70 ATOM 319 CG HIS 714 30.235 15.456 9.001 1.00 22.67 ATOM 320 CD2 HIS 714 30.626 16.751 9.026 1.00 22.73 ATOM 321 ND1 HIS 714 30.735 14.928 7.830 1.00 23.24 ATOM 322 CE1 HIS 714 31.401 15.863 7.175 1.00 22.99 ATOM 323 NE2 HIS 714 31.350 16.979 7.880 1.00 23.40 ATOM 324 C HIS 714 29.215 13.816 12.310 1.00 21.81 ATOM 325 O HIS 714 28.598 14.627 12.994 1.00 22.68 ATOM 326 N VAL 715 29.031 12.509 12.429 1.00 21.29 ATOM 327 CA VAL 715 28.099 11.992 13.423 1.00 20.49 ATOM 328 CB VAL 715 27.785 10.476 13.188 1.00 20.64 ATOM 329 CG1 VAL 715 27.000 9.897 14.361 1.00 20.52 ATOM 330 CG2 VAL 715 26.960 10.318 11.921 1.00 19.86 ATOM 331 C VAL 715 28.697 12.211 14.808 1.00 20.28 ATOM 332 O VAL 715 27.975 12.476 15.759 1.00 20.31 ATOM 333 N VAL 716 30.020 12.119 14.917 1.00 19.96 ATOM 334 CA VAL 716 30.680 12.331 16.203 1.00 19.21 ATOM 335 CB VAL 716 32.214 12.047 16.125 1.00 18.74 ATOM 336 CG1 VAL 716 32.889 12.440 17.427 1.00 17.72 ATOM 337 CG2 VAL 716 32.457 10.566 15.849 1.00 18.72 ATOM 338 C VAL 716 30.463 13.766 16.693 1.00 19.14 ATOM 339 O VAL 716 30.008 13.976 17.806 1.00 19.96 ATOM 340 N LYS 717 30.788 14.748 15.865 1.00 18.52 ATOM 341 CA LYS 717 30.620 16.139 16.256 1.00 18.43 ATOM 342 CB LYS 717 31.233 17.074 15.181 1.00 19.20 ATOM 343 CG LYS 717 32.760 17.074 15.191 1.00 20.49 ATOM 344 CD LYS 717 33.397 17.385 13.835 1.00 22.01 ATOM 345 CB LYS 717 33.242 18.841 13.423 1.00 23.42 ATOM 346 NZ LYS 717 34.179 19.195 12.301 1.00 23.88 ATOM 347 C LYS 717 29.149 16.462 16.484 1.00 17.83 ATOM 348 O LYS 717 28.809 17.280 17.330 1.00 18.37 ATOM 349 N TRP 718 28.276 15.806 15.733 1.00 17.26 ATOM 350 CA TRP 718 26.844 16.032 15.863 1.00 16.62 ATOM 351 CB TRP 718 26.096 15.283 14.747 1.00 15.39 ATOM 352 CG TRP 718 24.636 15.139 14.991 1.00 13.87 ATOM 353 CD2 TRP 718 23.955 13.949 15.416 1.00 13.60 ATOM 354 CE2 TRP 718 22.583 14.272 15.530 1.00 13.27 ATOM 355 CE3 TRP 718 24.373 12.639 15.710 1.00 12.87 ATOM 356 CD1 TRP 718 23.683 16.105 14.871 1.00 13.87 ATOM 357 NE1 TRP 718 22.444 15.594 15.191 1.00 13.38 ATOM 358 CZ2 TRP 718 21.622 13.334 15.929 1.00 12.65 ATOM 359 CZ3 TRP 718 23.417 11.705 16.106 1.00 12.99 ATOM 360 CH2 TRP 718 22.054 12.060 16.212 1.00 12.90 ATOM 361 C TRP 718 26.378 15.565 17.243 1.00 16.98 ATOM 362 O TRP 718 25.760 16.319 18.012 1.00 15.96 ATOM 363 N ALA 719 26.692 14.312 17.549 1.00 17.83 ATOM 364 CA ALA 719 26.326 13.721 18.819 1.00 18.17 ATOM 365 CB ALA 719 26.835 12.287 18.887 1.00 18.35 ATOM 366 C ALA 719 26.891 14.534 19.978 1.00 18.72 ATOM 367 O ALA 719 26.148 14.907 20.886 1.00 18.90 ATOM 368 N LYS 720 28.195 14.822 19.933 1.00 19.47 ATOM 369 CA LYS 720 28.870 15.570 21.003 1.00 20.52 ATOM 370 CB LYS 720 30.358 15.793 20.663 1.00 21.24 ATOM 371 CG LYS 720 31.257 14.541 20.709 1.00 21.82 ATOM 372 CD LYS 720 31.657 14.170 22.135 1.00 22.50 ATOM 373 CE LYS 720 32.458 12.850 22.217 1.00 23.14 ATOM 374 NZ LYS 720 33.777 12.847 21.507 1.00 22.87 ATOM 375 C LYS 720 28.215 16.912 21.316 1.00 20.70 ATOM 376 O LYS 720 28.338 17.418 22.429 1.00 21.08 ATOM 377 N ALA 721 27.520 17.487 20.338 1.00 20.77 ATOM 378 CA ALA 721 26.844 18.760 20.549 1.00 20.21 ATOM 379 CB ALA 721 26.944 19.614 19.300 1.00 20.14 ATOM 380 C ALA 721 25.378 18.592 20.944 1.00 20.23 ATOM 381 O ALA 721 24.664 19.582 21.088 1.00 20.39 ATOM 382 N LEU 722 24.925 17.352 21.116 1.00 20.40 ATOM 383 CA LEU 722 23.536 17.103 21.497 1.00 21.36 ATOM 384 CB LEU 722 23.166 15.627 21.282 1.00 21.25 ATOM 385 CG LEU 722 22.682 15.273 19.866 1.00 21.90 ATOM 386 CD1 LEU 722 22.656 13.756 19.658 1.00 20.78 ATOM 387 CD2 LEU 722 21.312 15.893 19.632 1.00 21.16 ATOM 388 C LEU 722 23.228 17.497 22.944 1.00 22.09 ATOM 389 O LEU 722 24.085 17.429 23.826 1.00 22.42 ATOM 390 N PRO 723 21.996 17.941 23.202 1.00 22.76 ATOM 391 CD PRO 723 20.936 18.384 22.273 1.00 22.83 ATOM 392 CA PRO 723 21.688 18.318 24.586 1.00 23.30 ATOM 393 CB PRO 723 20.256 18.860 24.490 1.00 23.27 ATOM 394 CG PRO 723 20.207 19.447 23.086 1.00 22.63 ATOM 395 C PRO 723 21.787 17.131 25.539 1.00 23.84 ATOM 396 O PRO 723 21.100 16.132 25.360 1.00 23.75 ATOM 397 N GLY 724 22.658 17.236 26.537 1.00 24.31 ATOM 398 CA GLY 724 22.788 16.172 27.524 1.00 24.68 ATOM 399 C GLY 724 23.653 14.966 27.180 1.00 25.55 ATOM 400 O GLY 724 23.896 14.114 28.040 1.00 25.10 ATOM 401 N PHE 725 24.113 14.876 25.935 1.00 25.87 ATOM 402 CA PHE 725 24.950 13.757 25.535 1.00 26.37 ATOM 403 CB PHE 725 25.373 13.898 24.077 1.00 25.67 ATOM 404 CG PHE 725 26.116 12.706 23.560 1.00 25.20 ATOM 405 CD1 PHE 725 25.428 11.572 23.140 1.00 24.92 ATOM 406 CD2 PHE 725 27.505 12.689 23.546 1.00 24.47 ATOM 407 CE1 PHE 725 26.117 10.429 22.712 1.00 24.76 ATOM 408 CE2 PHE 725 28.198 11.554 23.121 1.00 24.71 ATOM 409 CZ PHE 725 27.498 10.422 22.704 1.00 24.40 ATOM 410 C PHE 725 26.202 13.693 26.412 1.00 27.46 ATOM 411 O PHE 725 26.593 12.622 26.880 1.00 27.31 ATOM 412 N ARG 726 26.830 14.851 26.618 1.00 28.62 ATOM 413 CA ARG 726 28.039 14.951 27.430 1.00 29.72 ATOM 414 CB ARG 726 28.557 16.389 27.416 1.00 30.89 ATOM 415 CG ARG 726 29.272 16.781 26.134 1.00 32.52 ATOM 416 CD ARG 726 30.536 15.963 25.976 1.00 34.40 ATOM 417 NE ARG 726 31.176 15.731 27.271 1.00 35.49 ATOM 418 CZ ARG 726 32.382 15.196 27.428 1.00 36.20 ATOM 419 NH1 ARG 726 32.878 15.024 28.649 1.00 36.46 ATOM 420 NH2 ARG 726 33.098 14.846 26.365 1.00 36.56 ATOM 421 C ARG 726 27.855 14.480 28.879 1.00 29.93 ATOM 422 O ARG 726 28.825 14.378 29.636 1.00 29.51 ATOM 423 N ASN 727 26.613 14.212 29.274 1.00 29.89 ATOM 424 CA ASN 727 26.374 13.717 30.616 1.00 30.24 ATOM 425 CB ASN 727 24.891 13.782 30.968 1.00 30.69 ATOM 426 CG ASN 727 24.511 15.087 31.645 1.00 31.99 ATOM 427 OD1 ASN 727 23.357 15.539 31.562 1.00 32.19 ATOM 428 ND2 ASN 727 25.479 15.699 32.339 1.00 32.26 ATOM 429 C ASN 727 26.856 12.279 30.644 1.00 30.26 ATOM 430 O ASN 727 27.412 11.835 31.632 1.00 30.96 ATOM 431 N LEU 728 26.659 11.562 29.541 1.00 30.22 ATOM 432 CA LEU 728 27.080 10.167 29.444 1.00 30.16 ATOM 433 CB LEU 728 26.884 9.629 28.020 1.00 29.53 ATOM 434 CG LEU 728 25.469 9.476 27.448 1.00 29.44 ATOM 435 CD1 LEU 728 25.558 9.204 25.960 1.00 29.24 ATOM 436 CD2 LEU 728 24.738 8.351 28.144 1.00 29.16 ATOM 437 C LEU 728 28.545 10.049 29.800 1.00 30.48 ATOM 438 O LEU 728 29.315 10.995 29.624 1.00 30.21 ATOM 439 N HIS 729 28.925 8.884 30.302 1.00 30.85 ATOM 440 CA HIS 729 30.310 8.638 30.643 1.00 31.47 ATOM 441 CB HIS 729 30.453 7.268 31.327 1.00 31.98 ATOM 442 CG HIS 729 31.872 6.884 31.611 1.00 32.66 ATOM 443 CD2 HIS 729 32.844 6.406 30.796 1.00 32.89 ATOM 444 ND1 HIS 729 32.461 7.060 32.845 1.00 33.17 ATOM 445 CE1 HIS 729 33.735 6.713 32.777 1.00 33.16 ATOM 446 NE2 HIS 729 33.994 6.314 31.545 1.00 33.32 ATOM 447 C HIS 729 31.105 8.650 29.326 1.00 31.63 ATOM 448 O HIS 729 30.583 8.279 28.273 1.00 31.40 ATOM 449 N VAL 730 32.359 9.078 29.400 1.00 31.76 ATOM 450 CA VAL 730 33.250 9.133 28.250 1.00 32.24 ATOM 451 CB VAL 730 34.731 9.282 28.713 1.00 32.50 ATOM 452 CG1 VAL 730 35.658 9.405 27.508 1.00 32.05 ATOM 453 CG2 VAL 730 34.872 10.489 29.632 1.00 32.57 ATOM 454 C VAL 730 33.161 7.860 27.408 1.00 33.08 ATOM 455 O VAL 730 33.019 7.919 26.181 1.00 33.09 ATOM 456 N ASP 731 33.268 6.715 28.085 1.00 33.39 ATOM 457 CA ASP 731 33.246 5.400 27.449 1.00 33.76 ATOM 458 CB ASP 731 33.558 4.322 28.491 1.00 34.69 ATOM 459 CG ASP 731 34.969 4.433 29.033 1.00 36.10 ATOM 460 OD1 ASP 731 35.190 4.055 30.214 1.00 36.28 ATOM 461 OD2 ASP 731 35.856 4.892 28.270 1.00 36.30 ATOM 462 C ASP 731 31.946 5.054 26.738 1.00 33.23 ATOM 463 O ASP 731 31.971 4.444 25.674 1.00 33.20 ATOM 464 N ASP 732 30.822 5.428 27.344 1.00 32.63 ATOM 465 CA ASP 732 29.502 5.172 26.784 1.00 31.98 ATOM 466 CB ASP 732 28.418 5.345 27.861 1.00 32.27 ATOM 467 CG ASP 732 28.566 4.358 29.018 1.00 32.72 ATOM 468 OD1 ASP 732 29.043 3.223 28.783 1.00 33.04 ATOM 469 OD2 ASP 732 28.185 4.711 30.159 1.00 32.04 ATOM 470 C ASP 732 29.203 6.107 25.606 1.00 31.68 ATOM 471 O ASP 732 28.410 5.768 24.722 1.00 31.75 ATOM 472 N GLN 733 29.826 7.285 25.604 1.00 30.91 ATOM 473 CA GLN 733 29.631 8.249 24.526 1.00 30.18 ATOM 474 CB GLN 733 30.481 9.514 24.740 1.00 30.21 ATOM 475 CG GLN 733 30.198 10.316 25.996 1.00 31.08 ATOM 476 CD GLN 733 31.070 11.577 26.086 1.00 32.11 ATOM 477 OE1 GLN 733 32.282 11.528 25.855 1.00 33.33 ATOM 478 NE2 GLN 733 30.458 12.699 26.429 1.00 31.97 ATOM 479 C GLN 733 30.049 7.610 23.202 1.00 29.54 ATOM 480 O GLN 733 29.263 7.553 22.258 1.00 28.86 ATOM 481 N MET 734 31.287 7.124 23.146 1.00 29.26 ATOM 482 CA MET 734 31.818 6.505 21.929 1.00 29.62 ATOM 483 CB MET 734 33.347 6.432 21.993 1.00 31.24 ATOM 484 CG MET 734 34.044 7.704 21.512 1.00 34.06 ATOM 485 SD MET 734 33.906 8.000 19.707 1.00 37.92 ATOM 486 CE MET 734 35.327 7.026 19.055 1.00 36.58 ATOM 487 C MET 734 31.253 5.124 21.588 1.00 28.35 ATOM 488 O MET 734 31.280 4.722 20.428 1.00 28.45 ATOM 489 N ALA 735 30.765 4.398 22.589 1.00 26.73 ATOM 490 CA ALA 735 30.181 3.081 22.347 1.00 26.10 ATOM 491 CB ALA 735 30.003 2.310 23.669 1.00 26.35 ATOM 492 C ALA 735 28.825 3.298 21.676 1.00 24.76 ATOM 493 O ALA 735 28.531 2.705 20.653 1.00 24.32 ATOM 494 N VAL 736 28.012 4.160 22.272 1.00 24.18 ATOM 495 CA VAL 736 26.704 4.487 21.734 1.00 23.42 ATOM 496 CB VAL 736 26.022 5.579 22.592 1.00 23.57 ATOM 497 CG1 VAL 736 25.268 6.570 21.713 1.00 23.51 ATOM 498 CG2 VAL 736 25.071 4.932 23.573 1.00 23.24 ATOM 499 C VAL 736 26.895 4.985 20.301 1.00 23.21 ATOM 500 O VAL 736 26.228 4.516 19.375 1.00 23.40 ATOM 501 N ILE 737 27.827 5.917 20.129 1.00 22.03 ATOM 502 CA ILE 737 28.117 6.479 18.816 1.00 21.62 ATOM 503 CB ILE 737 29.228 7.550 18.883 1.00 20.18 ATOM 504 CG2 ILE 737 29.787 7.795 17.510 1.00 20.01 ATOM 505 CG1 ILE 737 28.685 8.855 19.468 1.00 19.45 ATOM 506 CD1 ILE 737 29.770 9.894 19.681 1.00 18.31 ATOM 507 C ILE 737 28.550 5.428 17.795 1.00 22.21 ATOM 508 O ILE 737 28.040 5.403 16.678 1.00 22.12 ATOM 509 N GLN 738 29.502 4.576 18.161 1.00 22.07 ATOM 510 CA GLN 738 29.949 3.572 17.225 1.00 22.29 ATOM 511 CB GLN 738 31.255 2.943 17.701 1.00 23.58 ATOM 512 CG GLN 738 32.411 3.940 17.806 1.00 23.70 ATOM 513 CD GLN 738 33.698 3.287 18.280 1.00 23.85 ATOM 514 OE1 GLN 738 33.682 2.436 19.165 1.00 24.48 ATOM 515 NE2 GLN 738 34.819 3.695 17.702 1.00 23.64 ATOM 516 C GLN 738 28.891 2.499 16.970 1.00 21.96 ATOM 517 O GLN 738 28.771 2.037 15.838 1.00 22.01 ATOM 518 N TYR 739 28.112 2.125 17.990 1.00 20.77 ATOM 519 CA TYR 739 27.073 1.115 17.800 1.00 19.98 ATOM 520 CB TYR 739 26.468 0.627 19.120 1.00 20.07 ATOM 521 CG TYR 739 27.407 0.014 20.133 1.00 20.08 ATOM 522 CD1 TYR 739 28.400 −0.886 19.754 1.00 20.37 ATOM 523 CE1 TYR 739 29.221 −1.484 20.703 1.00 21.09 ATOM 524 CD2 TYR 739 27.254 0.295 21.485 1.00 19.66 ATOM 525 CE2 TYR 739 28.065 −0.301 22.444 1.00 20.66 ATOM 526 CZ TYR 739 29.050 −1.182 22.049 1.00 20.98 ATOM 527 OH TYR 739 29.902 −1.706 22.998 1.00 22.42 ATOM 528 C TYR 739 25.897 1.621 16.964 1.00 19.75 ATOM 529 O TYR 739 25.249 0.845 16.250 1.00 19.75 ATOM 530 N SER 740 25.592 2.910 17.063 1.00 18.92 ATOM 531 CA SER 740 24.450 3.432 16.316 1.00 18.02 ATOM 532 CB SER 740 23.648 4.420 17.181 1.00 18.07 ATOM 533 OG SER 740 24.292 5.666 17.302 1.00 18.74 ATOM 534 C SER 740 24.831 4.069 14.984 1.00 16.97 ATOM 535 O SER 740 23.969 4.442 14.198 1.00 16.64 ATOM 536 N TRP 741 26.129 4.150 14.741 1.00 16.05 ATOM 537 CA TRP 741 26.689 4.725 13.529 1.00 16.05 ATOM 538 CB TRP 741 28.189 4.350 13.492 1.00 17.09 ATOM 539 CG TRP 741 29.006 4.764 12.306 1.00 19.02 ATOM 540 CD2 TRP 741 30.294 4.238 11.932 1.00 20.22 ATOM 541 CE2 TRP 741 30.662 4.848 10.713 1.00 20.50 ATOM 542 CE3 TRP 741 31.170 3.302 12.512 1.00 20.91 ATOM 543 CD1 TRP 741 28.667 5.657 11.335 1.00 19.78 ATOM 544 NE1 TRP 741 29.657 5.711 10.368 1.00 20.54 ATOM 545 CZ2 TRP 741 31.864 4.554 10.057 1.00 22.12 ATOM 546 CZ3 TRP 741 32.363 3.006 11.865 1.00 21.59 ATOM 547 CH2 TRP 741 32.702 3.632 10.643 1.00 22.46 ATOM 548 C TRP 741 25.911 4.300 12.257 1.00 15.45 ATOM 549 O TRP 741 25.401 5.150 11.526 1.00 14.12 ATOM 550 N MET 742 25.791 2.998 12.020 1.00 15.44 ATOM 551 CA MET 742 25.090 2.466 10.845 1.00 15.95 ATOM 552 CB MET 742 25.100 0.932 10.863 1.00 16.16 ATOM 553 CG MET 742 24.447 0.326 9.646 1.00 16.45 ATOM 554 SD MET 742 25.477 0.591 8.187 1.00 17.11 ATOM 555 CE MET 742 26.513 −0.885 8.352 1.00 16.44 ATOM 556 C MET 742 23.635 2.924 10.665 1.00 16.26 ATOM 557 O MET 742 23.259 3.451 9.597 1.00 16.31 ATOM 558 N GLY 743 22.826 2.683 11.698 1.00 16.23 ATOM 559 CA GLY 743 21.422 3.049 11.682 1.00 15.96 ATOM 560 C GLY 743 21.210 4.531 11.437 1.00 16.26 ATOM 561 O GLY 743 20.346 4.935 10.645 1.00 16.71 ATOM 562 N LEU 744 21.990 5.351 12.123 1.00 15.86 ATOM 563 CA LEU 744 21.888 6.791 11.952 1.00 15.37 ATOM 564 CB LEU 744 22.935 7.512 12.805 1.00 14.98 ATOM 565 CG LEU 744 22.689 7.629 14.308 1.00 15.65 ATOM 566 CD1 LEU 744 23.916 8.274 14.960 1.00 16.45 ATOM 567 CD2 LEU 744 21.447 8.482 14.581 1.00 14.77 ATOM 568 C LEU 744 22.100 7.146 10.483 1.00 15.33 ATOM 569 O LEU 744 21.350 7.939 9.918 1.00 14.53 ATOM 570 N MET 745 23.121 6.551 9.874 1.00 15.35 ATOM 571 CA MET 745 23.420 6.819 8.474 1.00 16.27 ATOM 572 CB MET 745 24.723 6.138 8.046 1.00 17.33 ATOM 573 CG MET 745 25.987 6.659 8.711 1.00 19.23 ATOM 574 SD MET 745 27.427 6.326 7.658 1.00 22.31 ATOM 575 CE MET 745 27.320 4.566 7.432 1.00 20.16 ATOM 576 C MET 745 22.297 6.350 7.540 1.00 16.24 ATOM 577 O MET 745 21.939 7.060 6.596 1.00 16.31 ATOM 578 N VAL 746 21.755 5.156 7.804 1.00 15.42 ATOM 579 CA VAL 746 20.685 4.615 6.981 1.00 14.36 ATOM 580 CB VAL 746 20.250 3.215 7.462 1.00 13.88 ATOM 581 CG1 VAL 746 18.936 2.830 6.808 1.00 13.61 ATOM 582 CG2 VAL 746 21.310 2.198 7.121 1.00 13.23 ATOM 583 C VAL 746 19.474 5.532 7.025 1.00 14.30 ATOM 584 O VAL 746 18.894 5.835 5.998 1.00 14.06 ATOM 585 N PHE 747 19.102 5.959 8.229 1.00 14.31 ATOM 586 CA PHE 747 17.953 6.831 8.433 1.00 14.49 ATOM 587 CB PHE 747 17.748 7.074 9.923 1.00 13.20 ATOM 588 CG PHE 747 16.425 7.677 10.263 1.00 12.24 ATOM 589 CD1 PHE 747 15.247 6.951 10.089 1.00 11.83 ATOM 590 CD2 PHE 747 16.348 8.954 10.802 1.00 12.05 ATOM 591 CE1 PHE 747 14.017 7.491 10.450 1.00 11.38 ATOM 592 CE2 PHE 747 15.120 9.498 11.168 1.00 11.97 ATOM 593 CZ PHE 747 13.951 8.765 10.991 1.00 10.82 ATOM 594 C PHE 747 18.094 8.180 7.715 1.00 15.24 ATOM 595 O PHE 747 17.192 8.590 6.994 1.00 15.13 ATOM 596 N ALA 748 19.211 8.870 7.930 1.00 15.97 ATOM 597 CA ALA 748 19.445 10.154 7.274 1.00 17.37 ATOM 598 CB ALA 748 20.765 10.746 7.728 1.00 15.48 ATOM 599 C ALA 748 19.460 9.940 5.758 1.00 18.41 ATOM 600 O ALA 748 18.875 10.702 4.998 1.00 19.17 ATOM 601 N MET 749 20.120 8.882 5.323 1.00 19.85 ATOM 602 CA MET 749 20.190 8.612 3.909 1.00 21.36 ATOM 603 CB MET 749 21.056 7.388 3.661 1.00 20.95 ATOM 604 CG MET 749 21.368 7.151 2.206 1.00 23.14 ATOM 605 SD MET 749 20.021 6.300 1.350 1.00 24.54 ATOM 606 CE MET 749 20.381 4.591 1.837 1.00 23.82 ATOM 607 C MET 749 18.762 8.415 3.395 1.00 22.45 ATOM 608 O MET 749 18.405 8.880 2.305 1.00 21.89 ATOM 609 N GLY 750 17.938 7.740 4.193 1.00 23.36 ATOM 610 CA GLY 750 16.562 7.522 3.787 1.00 23.69 ATOM 611 C GLY 750 15.934 8.874 3.507 1.00 24.41 ATOM 612 O GLY 750 15.329 9.091 2.451 1.00 23.64 ATOM 613 N TRP 751 16.097 9.793 4.461 1.00 25.13 ATOM 614 CA TRP 751 15.559 11.144 4.342 1.00 25.53 ATOM 615 CB TRP 751 15.937 11.957 5.570 1.00 25.52 ATOM 616 CG TRP 751 15.490 13.393 5.533 1.00 24.81 ATOM 617 CD2 TRP 751 14.156 13.886 5.715 1.00 24.48 ATOM 618 CE2 TRP 751 14.223 15.295 5.669 1.00 24.36 ATOM 619 CE3 TRP 751 12.911 13.273 5.913 1.00 24.15 ATOM 620 CD1 TRP 751 16.283 14.488 5.380 1.00 24.77 ATOM 621 NE1 TRP 751 15.532 15.635 5.464 1.00 24.45 ATOM 622 CZ2 TRP 751 13.096 16.104 5.816 1.00 24.20 ATOM 623 CZ3 TRP 751 11.794 14.072 6.058 1.00 24.68 ATOM 624 CH2 TRP 751 11.893 15.481 6.009 1.00 24.71 ATOM 625 C TRP 751 16.068 11.846 3.085 1.00 26.37 ATOM 626 O TRP 751 15.279 12.381 2.308 1.00 26.71 ATOM 627 N ARG 752 17.384 11.850 2.890 1.00 27.04 ATOM 628 CA ARG 752 17.963 12.478 1.713 1.00 27.72 ATOM 629 CB ARG 752 19.476 12.286 1.672 1.00 26.90 ATOM 630 CG ARG 752 20.229 13.200 2.598 1.00 25.98 ATOM 631 CD ARG 752 21.661 13.368 2.123 1.00 25.78 ATOM 632 NE ARG 752 22.336 12.086 1.971 1.00 24.53 ATOM 633 CZ ARG 752 22.620 11.278 2.986 1.00 24.29 ATOM 634 NH1 ARG 752 22.286 11.628 4.222 1.00 23.93 ATOM 635 NH2 ARG 752 23.237 10.127 2.764 1.00 23.51 ATOM 636 C ARG 752 17.373 11.933 0.427 1.00 28.66 ATOM 637 O ARG 752 17.107 12.694 −0.508 1.00 29.23 ATOM 638 N SER 753 17.169 10.620 0.367 1.00 29.64 ATOM 639 CA SER 753 16.612 10.015 −0.836 1.00 30.86 ATOM 640 CB SER 753 16.626 8.485 −0.739 1.00 30.75 ATOM 641 OG SER 753 17.950 7.982 −0.831 1.00 30.65 ATOM 642 C SER 753 15.196 10.508 −1.049 1.00 31.73 ATOM 643 O SER 753 14.681 10.498 −2.162 1.00 31.89 ATOM 644 N PHE 754 14.578 10.951 0.034 1.00 33.09 ATOM 645 CA PHE 754 13.220 11.457 −0.006 1.00 34.65 ATOM 646 CB PHE 754 12.604 11.394 1.391 1.00 34.66 ATOM 647 CG PHE 754 11.246 12.019 1.478 1.00 35.49 ATOM 648 CD1 PHE 754 10.165 11.463 0.799 1.00 35.64 ATOM 649 CD2 PHE 754 11.051 13.185 2.209 1.00 35.56 ATOM 650 CE1 PHE 754 8.913 12.061 0.845 1.00 35.75 ATOM 651 CE2 PHE 754 9.802 13.792 2.264 1.00 35.98 ATOM 652 CZ PHE 754 8.732 13.232 1.581 1.00 36.01 ATOM 653 C PHE 754 13.154 12.899 −0.519 1.00 35.79 ATOM 654 O PHE 754 12.448 13.193 −1.481 1.00 35.70 ATOM 655 N THR 755 13.896 13.789 0.133 1.00 37.07 ATOM 656 CA THR 755 13.904 15.204 −0.219 1.00 38.07 ATOM 657 CB THR 755 14.561 16.051 0.890 1.00 37.76 ATOM 658 OG1 THR 755 15.981 15.846 0.877 1.00 38.00 ATOM 659 CG2 THR 755 14.019 15.656 2.243 1.00 37.83 ATOM 660 C THR 755 14.626 15.516 −1.521 1.00 38.94 ATOM 661 O THR 755 14.855 16.684 −1.834 1.00 39.37 ATOM 662 N ASN 756 14.995 14.487 −2.275 1.00 39.70 ATOM 663 CA ASN 756 15.693 14.712 −3.533 1.00 40.27 ATOM 664 CB ASN 756 17.194 14.427 −3.369 1.00 40.38 ATOM 665 CG ASN 756 17.888 15.424 −2.434 1.00 40.95 ATOM 666 OD1 ASN 756 17.610 15.469 −1.237 1.00 41.37 ATOM 667 ND2 ASN 756 18.792 16.229 −2.986 1.00 41.31 ATOM 668 C ASN 756 15.128 13.904 −4.702 1.00 40.62 ATOM 669 O ASN 756 14.494 14.460 −5.599 1.00 40.90 ATOM 670 N VAL 757 15.337 12.594 −4.687 1.00 41.04 ATOM 671 CA VAL 757 14.869 11.743 −5.776 1.00 41.30 ATOM 672 CB VAL 757 15.939 10.680 −6.104 1.00 41.48 ATOM 673 CG1 VAL 757 17.218 11.363 −6.562 1.00 41.53 ATOM 674 CG2 VAL 757 16.226 9.835 −4.878 1.00 41.39 ATOM 675 C VAL 757 13.529 11.051 −5.518 1.00 41.34 ATOM 676 O VAL 757 13.206 10.046 −6.154 1.00 41.09 ATOM 677 N ASN 758 12.747 11.597 −4.593 1.00 41.49 ATOM 678 CA ASN 758 11.455 11.012 −4.246 1.00 41.40 ATOM 679 CB ASN 758 10.433 11.290 −5.346 1.00 41.86 ATOM 680 CG ASN 758 9.848 12.682 −5.251 1.00 42.34 ATOM 681 OD1 ASN 758 9.032 12.968 −4.370 1.00 42.45 ATOM 682 ND2 ASN 758 10.269 13.561 −6.153 1.00 42.55 ATOM 683 C ASN 758 11.548 9.511 −3.993 1.00 41.14 ATOM 684 O ASN 758 10.546 8.794 −4.062 1.00 41.19 ATOM 685 N SER 759 12.760 9.043 −3.716 1.00 40.61 ATOM 686 CA SER 759 13.003 7.639 −3.418 1.00 40.58 ATOM 687 CB SER 759 11.925 7.113 −2.462 1.00 40.93 ATOM 688 OG SER 759 11.878 7.895 −1.277 1.00 41.37 ATOM 689 C SER 759 13.114 6.704 −4.616 1.00 40.19 ATOM 690 O SER 759 13.004 5.490 −4.459 1.00 40.32 ATOM 691 N ARG 760 13.320 7.255 −5.809 1.00 39.83 ATOM 692 CA ARG 760 13.482 6.412 −6.996 1.00 39.13 ATOM 693 CB ARG 760 13.278 7.220 −8.289 1.00 40.09 ATOM 694 CG ARG 760 13.929 6.590 −9.533 1.00 41.59 ATOM 695 CD ARG 760 13.150 6.877 −10.827 1.00 42.76 ATOM 696 NE ARG 760 13.856 6.452 −12.047 1.00 44.10 ATOM 697 CZ ARG 760 14.317 5.221 −12.290 1.00 44.02 ATOM 698 NH1 ARG 760 14.169 4.244 −11.402 1.00 44.29 ATOM 699 NH2 ARG 760 14.926 4.966 −13.437 1.00 44.11 ATOM 700 C ARG 760 14.897 5.843 −6.951 1.00 37.86 ATOM 701 O ARG 760 15.200 4.841 −7.603 1.00 37.94 ATOM 702 N MET 761 15.760 6.489 −6.169 1.00 36.18 ATOM 703 CA MET 761 17.144 6.040 −6.030 1.00 34.54 ATOM 704 CB MET 761 18.026 6.693 −7.095 1.00 34.81 ATOM 705 CG MET 761 17.649 6.378 −8.531 1.00 35.67 ATOM 706 SD MET 761 18.836 7.089 −9.710 1.00 35.66 ATOM 707 CE MET 761 18.291 8.796 −9.734 1.00 35.50 ATOM 708 C MET 761 17.727 6.352 −4.652 1.00 32.75 ATOM 709 O MET 761 17.347 7.329 −4.009 1.00 32.64 ATOM 710 N LEU 762 18.661 5.521 −4.211 1.00 30.58 ATOM 711 CA LEU 762 19.309 5.728 −2.924 1.00 28.84 ATOM 712 CB LEU 762 19.967 4.435 −2.457 1.00 28.33 ATOM 713 CG LEU 762 19.019 3.248 −2.303 1.00 28.15 ATOM 714 CD1 LEU 762 19.810 2.045 −1.847 1.00 28.12 ATOM 715 CD2 LEU 762 17.924 3.575 −1.308 1.00 27.94 ATOM 716 C LEU 762 20.356 6.838 −3.040 1.00 27.76 ATOM 717 O LEU 762 21.341 6.713 −3.779 1.00 27.12 ATOM 718 N TYR 763 20.135 7.921 −2.302 1.00 26.72 ATOM 719 CA TYR 763 21.041 9.071 −2.318 1.00 26.03 ATOM 720 CB TYR 763 20.224 10.355 −2.181 1.00 26.73 ATOM 721 CG TYR 763 20.832 11.570 −2.836 1.00 28.00 ATOM 722 CD1 TYR 763 20.532 11.894 −4.159 1.00 28.78 ATOM 723 CE1 TYR 763 21.064 13.037 −4.764 1.00 29.17 ATOM 724 CD2 TYR 763 21.686 12.414 −2.131 1.00 28.81 ATOM 725 CE2 TYR 763 22.226 13.561 −2.729 1.00 29.70 ATOM 726 CZ TYR 763 21.907 13.865 −4.045 1.00 29.56 ATOM 727 OH TYR 763 22.410 15.005 −4.629 1.00 30.24 ATOM 728 C TYR 763 22.074 9.000 −1.180 1.00 25.05 ATOM 729 O TYR 763 21.886 9.607 −0.130 1.00 24.70 ATOM 730 N PHE 764 23.154 8.253 −1.386 1.00 23.85 ATOM 731 CA PHE 764 24.176 8.138 −0.363 1.00 23.36 ATOM 732 CB PHE 764 25.167 7.026 −0.687 1.00 22.20 ATOM 733 CG PHE 764 24.592 5.665 −0.577 1.00 20.93 ATOM 734 CD1 PHE 764 24.141 4.997 −1.703 1.00 21.03 ATOM 735 CD2 PHE 764 24.484 5.049 0.654 1.00 20.64 ATOM 736 CE1 PHE 764 23.590 3.728 −1.600 1.00 21.11 ATOM 737 CE2 PHE 764 23.936 3.785 0.765 1.00 20.71 ATOM 738 CZ PHE 764 23.489 3.123 −0.360 1.00 20.65 ATOM 739 C PHE 764 24.921 9.442 −0.246 1.00 23.54 ATOM 740 O PHE 764 25.270 9.866 0.848 1.00 23.77 ATOM 741 N ALA 765 25.154 10.067 −1.394 1.00 23.97 ATOM 742 CA ALA 765 25.854 11.346 −1.488 1.00 24.26 ATOM 743 CB ALA 765 27.346 11.142 −1.219 1.00 24.01 ATOM 744 C ALA 765 25.627 11.880 −2.909 1.00 24.66 ATOM 745 O ALA 765 25.211 11.130 −3.789 1.00 24.88 ATOM 746 N PRO 766 25.888 13.178 −3.153 1.00 25.03 ATOM 747 CD PRO 766 26.345 14.222 −2.220 1.00 25.22 ATOM 748 CA PRO 766 25.687 13.740 −4.496 1.00 25.14 ATOM 749 CB PRO 766 26.159 15.190 −4.335 1.00 25.71 ATOM 750 CG PRO 766 25.841 15.485 −2.888 1.00 25.20 ATOM 751 C PRO 766 26.485 12.980 −5.554 1.00 25.17 ATOM 752 O PRO 766 25.984 12.693 −6.651 1.00 25.08 ATOM 753 N ASP 767 27.721 12.634 −5.208 1.00 25.17 ATOM 754 CA ASP 767 28.600 11.898 −6.117 1.00 25.59 ATOM 755 CB ASP 767 30.058 12.292 −5.858 1.00 25.95 ATOM 756 CG ASP 767 30.531 11.898 −4.470 1.00 27.34 ATOM 757 OD1 ASP 767 29.780 12.093 −3.487 1.00 26.89 ATOM 758 OD2 ASP 767 31.668 11.400 −4.355 1.00 29.03 ATOM 759 C ASP 767 28.457 10.374 −6.012 1.00 25.16 ATOM 760 O ASP 767 29.215 9.644 −6.636 1.00 25.56 ATOM 761 N LEU 768 27.507 9.891 −5.214 1.00 24.75 ATOM 762 CA LEU 768 27.298 8.453 −5.076 1.00 24.23 ATOM 763 CB LEU 768 28.070 7.909 −3.870 1.00 23.92 ATOM 764 CG LEU 768 28.183 6.378 −3.771 1.00 23.88 ATOM 765 CD1 LEU 768 28.910 5.819 −4.974 1.00 23.45 ATOM 766 CD2 LEU 768 28.943 6.011 −2.523 1.00 24.21 ATOM 767 C LEU 768 25.808 8.129 −4.944 1.00 24.57 ATOM 768 O LEU 768 25.285 7.931 −3.846 1.00 24.30 ATOM 769 N VAL 769 25.133 8.081 −6.088 1.00 24.78 ATOM 770 CA VAL 769 23.702 7.802 −6.149 1.00 24.65 ATOM 771 CB VAL 769 22.998 8.807 −7.089 1.00 24.73 ATOM 772 CG1 VAL 769 21.480 8.621 −7.031 1.00 24.37 ATOM 773 CG2 VAL 769 23.398 10.245 −6.700 1.00 24.01 ATOM 774 C VAL 769 23.560 6.391 −6.698 1.00 24.90 ATOM 775 O VAL 769 24.156 6.061 −7.717 1.00 25.44 ATOM 776 N PHE 770 22.794 5.554 −6.007 1.00 24.72 ATOM 777 CA PHE 770 22.615 4.180 −6.434 1.00 24.40 ATOM 778 CB PHE 770 22.631 3.224 −5.243 1.00 24.46 ATOM 779 CG PHE 770 23.994 2.714 −4.867 1.00 23.68 ATOM 780 CD1 PHE 770 25.125 3.522 −4.993 1.00 22.58 ATOM 781 CD2 PHE 770 24.128 1.452 −4.294 1.00 23.29 ATOM 782 CE1 PHE 770 26.363 3.087 −4.552 1.00 22.46 ATOM 783 CE2 PHE 770 25.370 1.003 −3.841 1.00 23.62 ATOM 784 CZ PHE 770 26.497 1.833 −3.974 1.00 23.15 ATOM 785 C PHE 770 21.323 3.959 −7.167 1.00 24.70 ATOM 786 O PHE 770 20.237 4.104 −6.593 1.00 24.80 ATOM 787 N ASN 771 21.449 3.604 −8.438 1.00 24.57 ATOM 788 CA ASN 771 20.298 3.288 −9.259 1.00 24.55 ATOM 789 CB ASN 771 20.531 3.729 −10.699 1.00 24.45 ATOM 790 CG ASN 771 21.892 3.329 −11.216 1.00 24.66 ATOM 791 OD1 ASN 771 22.551 2.424 −10.678 1.00 24.49 ATOM 792 ND2 ASN 771 22.321 3.991 −12.277 1.00 24.40 ATOM 793 C ASN 771 20.195 1.770 −9.175 1.00 24.68 ATOM 794 O ASN 771 21.009 1.131 −8.510 1.00 24.39 ATOM 795 N GLU 772 19.211 1.185 −9.842 1.00 25.37 ATOM 796 CA GLU 772 19.052 −0.263 −9.790 1.00 25.97 ATOM 797 CB GLU 772 17.885 −0.698 −10.673 1.00 26.94 ATOM 798 CG GLU 772 16.519 −0.461 −10.013 1.00 28.24 ATOM 799 CD GLU 772 15.988 −1.704 −9.306 1.00 29.05 ATOM 800 OE1 GLU 772 15.347 −1.562 −8.235 1.00 29.60 ATOM 801 OE2 GLU 772 16.203 −2.819 −9.835 1.00 29.05 ATOM 802 C GLU 772 20.318 −0.987 −10.190 1.00 26.14 ATOM 803 O GLU 772 20.737 −1.934 −9.518 1.00 26.09 ATOM 804 N TYR 773 20.948 −0.530 −11.270 1.00 25.97 ATOM 805 CA TYR 773 22.166 −1.170 −11.734 1.00 25.56 ATOM 806 CB TYR 773 22.734 −0.445 −12.944 1.00 26.25 ATOM 807 CG TYR 773 24.033 −1.034 −13.428 1.00 25.78 ATOM 808 CD1 TYR 773 24.073 −2.281 −14.040 1.00 25.69 ATOM 809 CE1 TYR 773 25.282 −2.830 −14.469 1.00 26.68 ATOM 810 CD2 TYR 773 25.228 −0.344 −13.256 1.00 26.64 ATOM 811 CE2 TYR 773 26.448 −0.876 −13.683 1.00 26.53 ATOM 812 CZ TYR 773 26.469 −2.113 −14.286 1.00 26.86 ATOM 813 OH TYR 773 27.676 −2.624 −14.705 1.00 26.83 ATOM 814 C TYR 773 23.222 −1.234 −10.648 1.00 25.39 ATOM 815 O TYR 773 23.823 −2.286 −10.420 1.00 25.85 ATOM 816 N ARG 774 23.468 −0.117 −9.975 1.00 24.83 ATOM 817 CA ARG 774 24.467 −0.129 −8.913 1.00 24.13 ATOM 818 CB ARG 774 24.806 1.293 −8.493 1.00 23.53 ATOM 819 CG ARG 774 25.745 1.963 −9.465 1.00 23.97 ATOM 820 CD ARG 774 25.760 3.472 −9.277 1.00 24.07 ATOM 821 NE ARG 774 26.915 4.069 −9.934 1.00 24.01 ATOM 822 CZ ARG 774 27.280 5.332 −9.778 1.00 23.94 ATOM 823 NH1 ARG 774 26.576 6.124 −8.989 1.00 24.20 ATOM 824 NH2 ARG 774 28.350 5.797 −10.405 1.00 24.95 ATOM 825 C ARG 774 24.016 −0.965 −7.714 1.00 23.78 ATOM 826 O ARG 774 24.836 −1.553 −7.027 1.00 23.53 ATOM 827 N MET 775 22.714 −1.019 −7.470 1.00 23.55 ATOM 828 CA MET 775 22.189 −1.818 −6.375 1.00 24.24 ATOM 829 CB MET 775 20.680 −1.654 −6.298 1.00 23.35 ATOM 830 CG MET 775 20.279 −0.409 −5.602 1.00 22.56 ATOM 831 SD MET 775 18.562 −0.102 −5.803 1.00 23.81 ATOM 832 CE MET 775 18.596 1.595 −6.444 1.00 23.32 ATOM 833 C MET 775 22.544 −3.302 −6.556 1.00 25.19 ATOM 834 O MET 775 22.837 −4.011 −5.589 1.00 24.45 ATOM 835 N HIS 776 22.530 −3.757 −7.806 1.00 26.18 ATOM 836 CA HIS 776 22.845 −5.141 −8.119 1.00 26.92 ATOM 837 CB HIS 776 22.313 −5.505 −9.510 1.00 27.27 ATOM 838 CG HIS 776 22.615 −6.914 −9.913 1.00 28.49 ATOM 839 CD2 HIS 776 21.882 −8.049 −9.798 1.00 28.49 ATOM 840 ND1 HIS 776 23.841 −7.299 −10.415 1.00 28.47 ATOM 841 CE1 HIS 776 23.851 −8.611 −10.586 1.00 28.94 ATOM 842 NE2 HIS 776 22.675 −9.089 −10.219 1.00 29.04 ATOM 843 C HIS 776 24.344 −5.376 −8.070 1.00 27.70 ATOM 844 O HIS 776 24.818 −6.350 −7.481 1.00 28.14 ATOM 845 N LYS 777 25.086 −4.474 −8.699 1.00 28.16 ATOM 846 CA LYS 777 26.536 −4.555 −8.760 1.00 28.25 ATOM 847 CB LYS 777 27.050 −3.468 −9.702 1.00 28.87 ATOM 848 CG LYS 777 26.568 −3.632 −11.138 1.00 29.22 ATOM 849 CD LYS 777 27.637 −4.263 −12.030 1.00 29.26 ATOM 850 CE LYS 777 28.063 −5.643 −11.572 1.00 29.02 ATOM 851 NZ LYS 777 29.216 −6.132 −12.387 1.00 29.65 ATOM 852 C LYS 777 27.234 −4.440 −7.398 1.00 28.38 ATOM 853 O LYS 777 28.306 −5.006 −7.196 1.00 28.33 ATOM 854 N SER 778 26.638 −3.701 −6.468 1.00 28.47 ATOM 855 CA SER 778 27.227 −3.542 −5.143 1.00 28.59 ATOM 856 CB SER 778 26.574 −2.369 −4.408 1.00 28.41 ATOM 857 OG SER 778 25.199 −2.621 −4.172 1.00 27.42 ATOM 858 C SER 778 27.043 −4.820 −4.323 1.00 29.24 ATOM 859 O SER 778 27.756 −5.039 −3.352 1.00 29.39 ATOM 860 N ARG 779 26.078 −5.645 −4.733 1.00 29.69 ATOM 861 CA ARG 779 25.739 −6.904 −4.081 1.00 30.16 ATOM 862 CB ARG 779 26.998 −7.730 −3.817 1.00 30.77 ATOM 863 CG ARG 779 27.686 −8.208 −5.085 1.00 30.83 ATOM 864 CD ARG 779 28.959 −8.974 −4.796 1.00 31.77 ATOM 865 NE ARG 779 29.699 −9.207 −6.030 1.00 33.22 ATOM 866 CZ ARG 779 30.984 −9.547 −6.096 1.00 33.96 ATOM 867 NH1 ARG 779 31.700 −9.705 −4.987 1.00 33.90 ATOM 868 NH2 ARG 779 31.562 −9.699 −7.283 1.00 34.13 ATOM 869 C ARG 779 24.942 −6.698 −2.790 1.00 30.89 ATOM 870 O ARG 779 24.962 −7.541 −1.886 1.00 30.62 ATOM 871 N MET 780 24.232 −5.570 −2.729 1.00 31.21 ATOM 872 CA MET 780 23.384 −5.221 −1.592 1.00 31.28 ATOM 873 CB MET 780 23.948 −4.017 −0.832 1.00 32.57 ATOM 874 CG MET 780 25.288 −4.215 −0.172 1.00 34.02 ATOM 875 SD MET 780 25.814 −2.649 0.565 1.00 37.00 ATOM 876 CE MET 780 26.603 −1.893 −0.815 1.00 34.95 ATOM 877 C MET 780 22.010 −4.839 −2.137 1.00 30.81 ATOM 878 O MET 780 21.414 −3.841 −1.718 1.00 31.05 ATOM 879 N TYR 781 21.497 −5.621 −3.074 1.00 29.92 ATOM 880 CA TYR 781 20.208 −5.283 −3.651 1.00 29.23 ATOM 881 CB TYR 781 19.920 −6.156 −4.882 1.00 28.90 ATOM 882 CG TYR 781 18.721 −5.689 −5.670 1.00 28.52 ATOM 883 CD1 TYR 781 18.851 −4.762 −6.701 1.00 28.60 ATOM 884 CE1 TYR 781 17.726 −4.311 −7.408 1.00 28.68 ATOM 885 CD2 TYR 781 17.443 −6.152 −5.359 1.00 28.60 ATOM 886 CE2 TYR 781 16.318 −5.706 −6.051 1.00 28.33 ATOM 887 CZ TYR 781 16.461 −4.791 −7.071 1.00 28.53 ATOM 888 OH TYR 781 15.337 −4.364 −7.748 1.00 28.76 ATOM 889 C TYR 781 19.087 −5.407 −2.620 1.00 28.96 ATOM 890 O TYR 781 18.246 −4.513 −2.513 1.00 28.45 ATOM 891 N SER 782 19.068 −6.504 −1.862 1.00 28.78 ATOM 892 CA SER 782 18.030 −6.678 −0.835 1.00 28.74 ATOM 893 CB SER 782 18.209 −8.004 −0.100 1.00 29.02 ATOM 894 OG SER 782 18.036 −9.090 −0.987 1.00 30.66 ATOM 895 C SER 782 18.044 −5.531 0.187 1.00 27.95 ATOM 896 O SER 782 17.045 −4.839 0.363 1.00 28.09 ATOM 897 N GLN 783 19.170 −5.325 0.855 1.00 27.40 ATOM 898 CA GLN 783 19.249 −4.244 1.836 1.00 27.42 ATOM 899 CB GLN 783 20.669 −4.113 2.397 1.00 27.83 ATOM 900 CG GLN 783 21.268 −5.393 2.978 1.00 28.33 ATOM 901 CD GLN 783 21.844 −6.302 1.919 1.00 29.14 ATOM 902 OE1 GLN 783 22.658 −7.184 2.215 1.00 29.90 ATOM 903 NE2 GLN 783 21.426 −6.099 0.672 1.00 28.96 ATOM 904 C GLN 783 18.840 −2.924 1.177 1.00 26.96 ATOM 905 O GLN 783 18.124 −2.114 1.781 1.00 26.53 ATOM 906 N CYS 784 19.290 −2.719 −0.064 1.00 26.46 ATOM 907 CA CYS 784 18.963 −1.506 −0.808 1.00 26.18 ATOM 908 CB CYS 784 19.695 −1.488 −2.159 1.00 27.17 ATOM 909 SG CYS 784 21.458 −1.025 −2.047 1.00 28.41 ATOM 910 C CYS 784 17.452 −1.389 −1.002 1.00 25.21 ATOM 911 O CYS 784 16.901 −0.290 −0.952 1.00 24.95 ATOM 912 N VAL 785 16.789 −2.524 −1.210 1.00 24.67 ATOM 913 CA VAL 785 15.330 −2.554 −1.351 1.00 24.38 ATOM 914 CB VAL 785 14.821 −3.960 −1.738 1.00 24.36 ATOM 915 CG1 VAL 785 13.455 −4.230 −1.081 1.00 24.20 ATOM 916 CG2 VAL 785 14.705 −4.055 −3.247 1.00 23.60 ATOM 917 C VAL 785 14.698 −2.157 −0.021 1.00 23.98 ATOM 918 O VAL 785 13.705 −1.441 0.020 1.00 23.63 ATOM 919 N ARG 786 15.280 −2.646 1.067 1.00 24.16 ATOM 920 CA ARG 786 14.808 −2.304 2.411 1.00 24.19 ATOM 921 CB ARG 786 15.667 −3.016 3.465 1.00 24.76 ATOM 922 CG ARG 786 15.663 −4.537 3.386 1.00 25.63 ATOM 923 CD ARG 786 14.617 −5.109 4.314 1.00 26.26 ATOM 924 NE ARG 786 15.149 −6.186 5.141 1.00 26.47 ATOM 925 CZ ARG 786 14.769 −6.414 6.396 1.00 26.71 ATOM 926 NH1 ARG 786 13.861 −5.637 6.970 1.00 26.62 ATOM 927 NH2 ARG 786 15.283 −7.426 7.074 1.00 26.60 ATOM 928 C ARG 786 14.934 −0.783 2.613 1.00 23.52 ATOM 929 O ARG 786 14.015 −0.128 3.087 1.00 23.13 ATOM 930 N MET 787 16.079 −0.227 2.236 1.00 23.46 ATOM 931 CA MET 787 16.326 1.201 2.404 1.00 24.21 ATOM 932 CB MET 787 17.799 1.506 2.153 1.00 24.32 ATOM 933 CG MET 787 18.695 0.872 3.191 1.00 25.11 ATOM 934 SD MET 787 20.411 1.249 2.977 1.00 26.06 ATOM 935 CE MET 787 20.960 −0.200 2.082 1.00 25.69 ATOM 936 C MET 787 15.443 2.082 1.535 1.00 24.88 ATOM 937 O MET 787 14.947 3.113 1.999 1.00 24.39 ATOM 938 N ARG 788 15.260 1.675 0.278 1.00 25.61 ATOM 939 CA ARG 788 14.399 2.394 −0.651 1.00 26.64 ATOM 940 CB ARG 788 14.361 1.661 −2.003 1.00 27.51 ATOM 941 CG ARG 788 13.561 2.371 −3.089 1.00 28.69 ATOM 942 CD ARG 788 14.374 2.537 −4.378 1.00 29.41 ATOM 943 NE ARG 788 14.059 1.556 −5.419 1.00 29.78 ATOM 944 CZ ARG 788 14.634 1.548 −6.626 1.00 31.03 ATOM 945 NH1 ARG 788 15.549 2.465 −6.935 1.00 30.51 ATOM 946 NH2 ARG 788 14.298 0.631 −7.532 1.00 30.16 ATOM 947 C ARG 788 13.016 2.401 0.007 1.00 26.84 ATOM 948 O ARG 788 12.328 3.418 0.047 1.00 26.40 ATOM 949 N HIS 789 12.632 1.251 0.545 1.00 27.75 ATOM 950 CA HIS 789 11.359 1.104 1.233 1.00 29.39 ATOM 951 CB HIS 789 11.238 −0.300 1.811 1.00 30.70 ATOM 952 CG HIS 789 9.968 −0.996 1.447 1.00 32.08 ATOM 953 CD2 HIS 789 8.916 −1.387 2.205 1.00 32.40 ATOM 954 ND1 HIS 789 9.685 −1.396 0.157 1.00 32.78 ATOM 955 CE1 HIS 789 8.513 −2.009 0.139 1.00 33.40 ATOM 956 NE2 HIS 789 8.026 −2.017 1.369 1.00 33.45 ATOM 957 C HIS 789 11.275 2.120 2.373 1.00 29.90 ATOM 958 O HIS 789 10.274 2.829 2.513 1.00 29.93 ATOM 959 N LEU 790 12.328 2.173 3.192 1.00 30.02 ATOM 960 CA LEU 790 12.390 3.115 4.307 1.00 30.13 ATOM 961 CB LEU 790 13.775 3.071 4.956 1.00 30.30 ATOM 962 CG LEU 790 14.142 4.222 5.896 1.00 30.27 ATOM 963 CD1 LEU 790 13.147 4.322 7.029 1.00 30.03 ATOM 964 CD2 LEU 790 15.536 3.978 6.445 1.00 31.35 ATOM 965 C LEU 790 12.107 4.533 3.836 1.00 30.25 ATOM 966 O LEU 790 11.194 5.189 4.325 1.00 30.14 ATOM 967 N SER 791 12.911 5.000 2.890 1.00 30.62 ATOM 968 CA SER 791 12.759 6.334 2.338 1.00 31.31 ATOM 969 CB SER 791 13.661 6.504 1.123 1.00 31.61 ATOM 970 OG SER 791 13.224 5.654 0.082 1.00 32.91 ATOM 971 C SER 791 11.318 6.603 1.920 1.00 31.63 ATOM 972 O SER 791 10.747 7.626 2.290 1.00 32.03 ATOM 973 N GLN 792 10.734 5.696 1.138 1.00 31.71 ATOM 974 CA GLN 792 9.354 5.873 0.687 1.00 31.71 ATOM 975 CB GLN 792 8.909 4.666 −0.150 1.00 31.94 ATOM 976 CG GLN 792 9.637 4.593 −1.492 1.00 32.57 ATOM 977 CD GLN 792 9.418 3.282 −2.223 1.00 33.30 ATOM 978 OE1 GLN 792 8.385 3.073 −2.860 1.00 33.62 ATOM 979 NE2 GLN 792 10.394 2.384 −2.128 1.00 33.08 ATOM 980 C GLN 792 8.417 6.107 1.870 1.00 31.60 ATOM 981 O GLN 792 7.459 6.881 1.766 1.00 31.38 ATOM 982 N GLU 793 8.706 5.455 2.997 1.00 31.29 ATOM 983 CA GLU 793 7.913 5.629 4.216 1.00 30.78 ATOM 984 CB GLU 793 8.490 4.782 5.353 1.00 31.16 ATOM 985 CG GLU 793 8.422 3.301 5.099 1.00 31.63 ATOM 986 CD GLU 793 7.024 2.753 5.256 1.00 32.41 ATOM 987 OE1 GLU 793 6.056 3.549 5.178 1.00 32.89 ATOM 988 OE2 GLU 793 6.892 1.523 5.447 1.00 32.77 ATOM 989 C GLU 793 7.927 7.101 4.630 1.00 30.41 ATOM 990 O GLU 793 6.959 7.609 5.202 1.00 30.34 ATOM 991 N PHE 794 9.032 7.787 4.355 1.00 29.94 ATOM 992 CA PHE 794 9.119 9.195 4.703 1.00 29.89 ATOM 993 CB PHE 794 10.498 9.762 4.367 1.00 29.61 ATOM 994 CG PHE 794 11.586 9.331 5.316 1.00 29.83 ATOM 995 CD1 PHE 794 11.472 9.567 6.683 1.00 29.77 ATOM 996 CD2 PHE 794 12.722 8.684 4.848 1.00 29.90 ATOM 997 CE1 PHE 794 12.466 9.161 7.561 1.00 29.72 ATOM 998 CE2 PHE 794 13.722 8.274 5.726 1.00 30.02 ATOM 999 CZ PHE 794 13.592 8.513 7.083 1.00 29.58 ATOM 1000 C PHE 794 8.057 9.943 3.922 1.00 30.47 ATOM 1001 O PHE 794 7.721 11.075 4.248 1.00 30.71 ATOM 1002 N GLY 795 7.532 9.296 2.883 1.00 31.23 ATOM 1003 CA GLY 795 6.513 9.910 2.053 1.00 31.91 ATOM 1004 C GLY 795 5.101 9.482 2.410 1.00 32.71 ATOM 1005 O GLY 795 4.219 10.322 2.557 1.00 32.69 ATOM 1006 N TRP 796 4.875 8.181 2.557 1.00 33.17 ATOM 1007 CA TRP 796 3.539 7.701 2.897 1.00 34.04 ATOM 1008 CB TRP 796 3.490 6.176 2.820 1.00 33.54 ATOM 1009 CG TRP 796 3.880 5.642 1.484 1.00 33.47 ATOM 1010 CD2 TRP 796 4.706 4.499 1.223 1.00 33.26 ATOM 1011 CE2 TRP 796 4.779 4.347 −0.183 1.00 32.92 ATOM 1012 CE3 TRP 796 5.391 3.587 2.039 1.00 33.53 ATOM 1013 CD1 TRP 796 3.497 6.125 0.259 1.00 33.23 ATOM 1014 NE1 TRP 796 4.033 5.351 −0.743 1.00 32.89 ATOM 1015 CZ2 TRP 796 5.509 3.320 −0.793 1.00 32.88 ATOM 1016 CZ3 TRP 796 6.124 2.556 1.426 1.00 33.73 ATOM 1017 CH2 TRP 796 6.172 2.438 0.022 1.00 33.34 ATOM 1018 C TRP 796 3.079 8.168 4.283 1.00 34.95 ATOM 1019 O TRP 796 1.883 8.130 4.596 1.00 35.51 ATOM 1020 N LEU 797 4.026 8.620 5.105 1.00 35.59 ATOM 1021 CA LEU 797 3.712 9.097 6.451 1.00 35.60 ATOM 1022 CB LEU 797 4.672 8.487 7.466 1.00 35.55 ATOM 1023 CG LEU 797 4.539 6.978 7.667 1.00 35.46 ATOM 1024 CD1 LEU 797 5.745 6.465 8.443 1.00 35.46 ATOM 1025 CD2 LEU 797 3.232 6.671 8.390 1.00 35.11 ATOM 1026 C LEU 797 3.794 10.610 6.545 1.00 35.92 ATOM 1027 O LEU 797 3.574 11.182 7.612 1.00 36.29 ATOM 1028 N GLN 798 4.103 11.257 5.427 1.00 36.20 ATOM 1029 CA GLN 798 4.224 12.711 5.397 1.00 36.28 ATOM 1030 CB GLN 798 2.830 13.377 5.366 1.00 37.02 ATOM 1031 CG GLN 798 2.233 13.503 3.939 1.00 38.04 ATOM 1032 CD GLN 798 0.757 13.919 3.908 1.00 38.46 ATOM 1033 OE1 GLN 798 −0.113 13.231 4.458 1.00 39.04 ATOM 1034 NE2 GLN 798 0.472 15.036 3.247 1.00 38.17 ATOM 1035 C GLN 798 5.033 13.185 6.598 1.00 35.87 ATOM 1036 O GLN 798 4.637 14.099 7.321 1.00 36.50 ATOM 1037 N ILE 799 6.179 12.543 6.794 1.00 35.08 ATOM 1038 CA ILE 799 7.084 12.869 7.885 1.00 34.09 ATOM 1039 CB ILE 799 8.205 11.824 7.982 1.00 33.96 ATOM 1040 CG2 ILE 799 9.294 12.288 8.934 1.00 33.52 ATOM 1041 CG1 ILE 799 7.598 10.504 8.439 1.00 34.19 ATOM 1042 CD1 ILE 799 8.575 9.407 8.549 1.00 35.01 ATOM 1043 C ILE 799 7.691 14.247 7.694 1.00 33.51 ATOM 1044 O ILE 799 8.244 14.547 6.645 1.00 33.24 ATOM 1045 N THR 800 7.587 15.074 8.728 1.00 33.31 ATOM 1046 CA THR 800 8.102 16.436 8.700 1.00 33.05 ATOM 1047 CB THR 800 7.351 17.341 9.717 1.00 33.44 ATOM 1048 OG1 THR 800 7.803 17.043 11.048 1.00 33.14 ATOM 1049 CG2 THR 800 5.840 17.113 9.636 1.00 32.47 ATOM 1050 C THR 800 9.582 16.515 9.050 1.00 33.21 ATOM 1051 O THR 800 10.166 15.577 9.599 1.00 33.40 ATOM 1052 N PRO 801 10.214 17.644 8.719 1.00 33.07 ATOM 1053 CD PRO 801 9.726 18.642 7.748 1.00 33.01 ATOM 1054 CA PRO 801 11.630 17.858 9.007 1.00 32.90 ATOM 1055 CB PRO 801 11.891 19.229 8.386 1.00 32.72 ATOM 1056 CG PRO 801 11.012 19.196 7.189 1.00 32.68 ATOM 1057 C PRO 801 11.886 17.841 10.521 1.00 32.91 ATOM 1058 O PRO 801 12.899 17.314 10.995 1.00 33.12 ATOM 1059 N GLN 802 10.967 18.426 11.281 1.00 32.37 ATOM 1060 CA GLN 802 11.117 18.458 12.729 1.00 31.71 ATOM 1061 CB GLN 802 10.090 19.425 13.340 1.00 32.11 ATOM 1062 CG GLN 802 10.276 20.925 12.948 1.00 32.69 ATOM 1063 CD GLN 802 9.954 21.244 11.464 1.00 33.21 ATOM 1064 OE1 GLN 802 8.898 20.871 10.943 1.00 33.11 ATOM 1065 NE2 GLN 802 10.865 21.956 10.798 1.00 33.53 ATOM 1066 C GLN 802 10.987 17.045 13.338 1.00 31.06 ATOM 1067 O GLN 802 11.680 16.711 14.305 1.00 31.17 ATOM 1068 N GLU 803 10.109 16.217 12.775 1.00 29.88 ATOM 1069 CA GLU 803 9.935 14.849 13.269 1.00 28.79 ATOM 1070 CB GLU 803 8.750 14.154 12.587 1.00 29.29 ATOM 1071 CG GLU 803 7.378 14.677 12.936 1.00 30.44 ATOM 1072 CD GLU 803 6.286 13.977 12.145 1.00 30.78 ATOM 1073 OE1 GLU 803 6.277 14.099 10.905 1.00 29.94 ATOM 1074 OE2 GLU 803 5.440 13.295 12.766 1.00 32.29 ATOM 1075 C GLU 803 11.198 14.058 12.940 1.00 27.63 ATOM 1076 O GLU 803 11.743 13.358 13.783 1.00 27.67 ATOM 1077 N PHE 804 11.644 14.167 11.694 1.00 25.86 ATOM 1078 CA PHE 804 12.835 13.472 11.252 1.00 24.53 ATOM 1079 CB PHE 804 13.176 13.850 9.803 1.00 23.88 ATOM 1080 CG PHE 804 14.603 13.578 9.439 1.00 23.21 ATOM 1081 CD1 PHE 804 15.098 12.280 9.449 1.00 22.70 ATOM 1082 CD2 PHE 804 15.475 14.625 9.164 1.00 23.07 ATOM 1083 CE1 PHE 804 16.436 12.031 9.199 1.00 22.58 ATOM 1084 CE2 PHE 804 16.823 14.384 8.909 1.00 22.21 ATOM 1085 CZ PHE 804 17.303 13.091 8.929 1.00 22.63 ATOM 1086 C PHE 804 14.044 13.755 12.146 1.00 23.92 ATOM 1087 O PHE 804 14.797 12.841 12.480 1.00 23.49 ATOM 1088 N LEU 805 14.242 15.013 12.517 1.00 23.43 ATOM 1089 CA LEU 805 15.377 15.360 13.362 1.00 23.07 ATOM 1090 CB LEU 805 15.572 16.881 13.432 1.00 22.40 ATOM 1091 CG LEU 805 15.862 17.582 12.100 1.00 22.11 ATOM 1092 CD1 LEU 805 16.020 19.075 12.339 1.00 22.35 ATOM 1093 CD2 LEU 805 17.104 17.007 11.441 1.00 21.68 ATOM 1094 C LEU 805 15.240 14.791 14.761 1.00 23.01 ATOM 1095 O LEU 805 16.231 14.404 15.358 1.00 23.29 ATOM 1096 N CYS 806 14.028 14.731 15.301 1.00 23.32 ATOM 1097 CA CYS 806 13.872 14.151 16.638 1.00 23.74 ATOM 1098 CB CYS 806 12.469 14.379 17.179 1.00 24.34 ATOM 1099 SG CYS 806 12.141 16.076 17.527 1.00 30.40 ATOM 1100 C CYS 806 14.145 12.649 16.605 1.00 22.67 ATOM 1101 O CYS 806 14.877 12.117 17.447 1.00 22.58 ATOM 1102 N MET 807 13.543 11.971 15.637 1.00 21.64 ATOM 1103 CA MET 807 13.724 10.532 15.503 1.00 21.18 ATOM 1104 CB MET 807 12.919 9.991 14.313 1.00 22.00 ATOM 1105 CG MET 807 11.403 10.114 14.463 1.00 23.29 ATOM 1106 SD MET 807 10.507 9.386 13.019 1.00 25.08 ATOM 1107 CE MET 807 10.295 10.783 12.018 1.00 24.91 ATOM 1108 C MET 807 15.204 10.193 15.330 1.00 19.80 ATOM 1109 O MET 807 15.698 9.267 15.950 1.00 19.29 ATOM 1110 N LYS 808 15.900 10.950 14.488 1.00 18.78 ATOM 1111 CA LYS 808 17.321 10.730 14.250 1.00 18.18 ATOM 1112 CB LYS 808 17.877 11.789 13.284 1.00 17.70 ATOM 1113 CG LYS 808 19.376 11.652 13.044 1.00 18.55 ATOM 1114 CD LYS 808 19.775 11.948 11.583 1.00 18.83 ATOM 1115 CE LYS 808 19.711 13.425 11.271 1.00 17.98 ATOM 1116 NZ LYS 808 20.547 14.166 12.258 1.00 18.71 ATOM 1117 C LYS 808 18.063 10.786 15.588 1.00 17.76 ATOM 1118 O LYS 808 18.823 9.880 15.921 1.00 16.45 ATOM 1119 N ALA 809 17.829 11.853 16.347 1.00 17.98 ATOM 1120 CA ALA 809 18.443 12.011 17.661 1.00 18.61 ATOM 1121 CB ALA 809 17.950 13.283 18.321 1.00 18.30 ATOM 1122 C ALA 809 18.105 10.786 18.532 1.00 18.88 ATOM 1123 O ALA 809 18.986 10.210 19.167 1.00 18.79 ATOM 1124 N LEU 810 16.842 10.369 18.533 1.00 19.30 ATOM 1125 CA LEU 810 16.432 9.204 19.321 1.00 19.68 ATOM 1126 CB LEU 810 14.917 9.033 19.242 1.00 19.80 ATOM 1127 CG LEU 810 14.250 8.133 20.290 1.00 21.07 ATOM 1128 CD1 LEU 810 14.607 8.601 21.706 1.00 21.00 ATOM 1129 CD2 LEU 810 12.736 8.168 20.094 1.00 20.91 ATOM 1130 C LEU 810 17.137 7.924 18.837 1.00 19.90 ATOM 1131 O LEU 810 17.497 7.051 19.631 1.00 19.94 ATOM 1132 N LEU 811 17.347 7.824 17.530 1.00 20.06 ATOM 1133 CA LEU 811 18.010 6.665 16.949 1.00 20.23 ATOM 1134 CB LEU 811 18.125 6.825 15.424 1.00 19.95 ATOM 1135 CG LEU 811 17.498 5.720 14.561 1.00 20.82 ATOM 1136 CD1 LEU 811 17.661 6.059 13.079 1.00 20.39 ATOM 1137 CD2 LEU 811 18.139 4.380 14.874 1.00 20.09 ATOM 1138 C LEU 811 19.407 6.447 17.555 1.00 20.02 ATOM 1139 O LEU 811 19.889 5.326 17.622 1.00 20.87 ATOM 1140 N LEU 812 20.060 7.513 17.992 1.00 19.93 ATOM 1141 CA LEU 812 21.389 7.380 18.581 1.00 19.94 ATOM 1142 CB LEU 812 22.041 8.753 18.753 1.00 19.93 ATOM 1143 CG LEU 812 23.309 8.740 19.607 1.00 19.48 ATOM 1144 CD1 LEU 812 24.523 8.421 18.748 1.00 19.57 ATOM 1145 CD2 LEU 812 23.467 10.068 20.272 1.00 19.85 ATOM 1146 C LEU 812 21.355 6.692 19.940 1.00 19.79 ATOM 1147 O LEU 812 22.383 6.237 20.414 1.00 19.89 ATOM 1148 N PHE 813 20.182 6.638 20.564 1.00 20.27 ATOM 1149 CA PHE 813 20.025 6.011 21.872 1.00 20.89 ATOM 1150 CB PHE 813 19.334 6.988 22.830 1.00 21.71 ATOM 1151 CG PHE 813 20.049 8.309 22.964 1.00 22.53 ATOM 1152 CD1 PHE 813 21.296 8.381 23.575 1.00 22.94 ATOM 1153 CD2 PHE 813 19.483 9.475 22.452 1.00 22.60 ATOM 1154 CE1 PHE 813 21.974 9.600 23.676 1.00 23.67 ATOM 1155 CE2 PHE 813 20.143 10.698 22.543 1.00 23.12 ATOM 1156 CZ PHE 813 21.392 10.764 23.156 1.00 24.01 ATOM 1157 C PHE 813 19.233 4.706 21.814 1.00 21.29 ATOM 1158 O PHE 813 18.573 4.327 22.780 1.00 20.70 ATOM 1159 N SER 814 19.304 4.013 20.680 1.00 22.24 ATOM 1160 CA SER 814 18.571 2.762 20.526 1.00 22.82 ATOM 1161 CB SER 814 17.604 2.870 19.352 1.00 23.27 ATOM 1162 OG SER 814 16.529 3.731 19.689 1.00 25.04 ATOM 1163 C SER 814 19.402 1.502 20.387 1.00 22.78 ATOM 1164 O SER 814 18.901 0.486 19.933 1.00 23.31 ATOM 1165 N ILE 815 20.664 1.554 20.792 1.00 23.17 ATOM 1166 CA ILE 815 21.519 0.388 20.715 1.00 24.25 ATOM 1167 CB ILE 815 22.260 0.339 19.361 1.00 25.14 ATOM 1168 CG2 ILE 815 22.988 1.640 19.099 1.00 25.51 ATOM 1169 CG1 ILE 815 23.222 −0.849 19.343 1.00 26.20 ATOM 1170 CD1 ILE 815 22.524 −2.177 19.315 1.00 26.03 ATOM 1171 C ILE 815 22.500 0.376 21.888 1.00 24.71 ATOM 1172 O ILE 815 23.306 1.292 22.065 1.00 24.79 ATOM 1173 N ILE 816 22.417 −0.671 22.700 1.00 25.36 ATOM 1174 CA ILE 816 23.263 −0.784 23.887 1.00 26.22 ATOM 1175 CB ILE 816 22.595 −0.092 25.081 1.00 25.90 ATOM 1176 CG2 ILE 816 22.537 1.422 24.853 1.00 25.91 ATOM 1177 CG1 ILE 816 21.202 −0.690 25.282 1.00 25.62 ATOM 1178 CD1 ILE 816 20.460 −0.144 26.481 1.00 26.47 ATOM 1179 C ILE 816 23.570 −2.220 24.328 1.00 26.59 ATOM 1180 O ILE 816 22.807 −3.142 24.048 1.00 26.12 ATOM 1181 N PRO 817 24.694 −2.413 25.041 1.00 27.17 ATOM 1182 CD PRO 817 25.758 −1.427 25.304 1.00 27.29 ATOM 1183 CA PRO 817 25.085 −3.742 25.525 1.00 27.80 ATOM 1184 CB PRO 817 26.384 −3.474 26.284 1.00 27.15 ATOM 1185 CG PRO 817 26.958 −2.313 25.557 1.00 27.61 ATOM 1186 C PRO 817 24.010 −4.324 26.435 1.00 28.43 ATOM 1187 O PRO 817 23.380 −3.600 27.212 1.00 27.45 ATOM 1188 N VAL 818 23.808 −5.634 26.312 1.00 29.98 ATOM 1189 CA VAL 818 22.833 −6.380 27.108 1.00 31.48 ATOM 1190 CB VAL 818 22.947 −7.891 26.825 1.00 31.98 ATOM 1191 CG1 VAL 818 24.335 −8.377 27.215 1.00 33.11 ATOM 1192 CG2 VAL 818 21.879 −8.665 27.590 1.00 32.85 ATOM 1193 C VAL 818 23.032 −6.163 28.609 1.00 32.15 ATOM 1194 O VAL 818 22.069 −6.152 29.364 1.00 32.43 ATOM 1195 N ASP 819 24.276 −5.997 29.046 1.00 33.13 ATOM 1196 CA ASP 819 24.536 −5.786 30.466 1.00 34.13 ATOM 1197 CB ASP 819 25.707 −6.662 30.932 1.00 35.28 ATOM 1198 CG ASP 819 27.024 −6.302 30.258 1.00 36.52 ATOM 1199 OD1 ASP 819 27.991 −7.091 30.403 1.00 37.48 ATOM 1200 OD2 ASP 819 27.097 −5.242 29.591 1.00 37.03 ATOM 1201 C ASP 819 24.798 −4.315 30.798 1.00 34.30 ATOM 1202 O ASP 819 25.668 −3.989 31.610 1.00 33.84 ATOM 1203 N GLY 820 24.026 −3.440 30.150 1.00 34.60 ATOM 1204 CA GLY 820 24.125 −2.005 30.361 1.00 34.11 ATOM 1205 C GLY 820 25.477 −1.375 30.121 1.00 33.97 ATOM 1206 O GLY 820 26.474 −2.068 29.945 1.00 33.92 ATOM 1207 N LEU 821 25.493 −0.042 30.133 1.00 34.32 ATOM 1208 CA LEU 821 26.693 0.768 29.925 1.00 34.47 ATOM 1209 CB LEU 821 26.330 2.053 29.191 1.00 34.92 ATOM 1210 CG LEU 821 25.887 2.042 27.733 1.00 35.26 ATOM 1211 CD1 LEU 821 25.251 3.398 27.420 1.00 35.53 ATOM 1212 CD2 LEU 821 27.080 1.769 26.819 1.00 34.91 ATOM 1213 C LEU 821 27.348 1.154 31.242 1.00 34.74 ATOM 1214 O LEU 821 26.836 0.841 32.313 1.00 34.48 ATOM 1215 N LYS 822 28.476 1.855 31.148 1.00 35.36 ATOM 1216 CA LYS 822 29.211 2.316 32.325 1.00 36.27 ATOM 1217 CB LYS 822 30.384 3.204 31.903 1.00 36.87 ATOM 1218 CG LYS 822 31.286 2.590 30.856 1.00 37.44 ATOM 1219 CD LYS 822 31.934 1.317 31.361 1.00 38.01 ATOM 1220 CE LYS 822 32.921 0.770 30.343 1.00 38.61 ATOM 1221 NZ LYS 822 34.065 1.699 30.117 1.00 39.07 ATOM 1222 C LYS 822 28.296 3.112 33.263 1.00 36.51 ATOM 1223 O LYS 822 28.440 3.056 34.480 1.00 36.71 ATOM 1224 N ASN 823 27.359 3.859 32.690 1.00 36.69 ATOM 1225 CA ASN 823 26.424 4.644 33.483 1.00 36.49 ATOM 1226 CB ASN 823 26.893 6.098 33.529 1.00 37.62 ATOM 1227 CG ASN 823 28.271 6.239 34.150 1.00 39.08 ATOM 1228 OD1 ASN 823 29.251 5.668 33.658 1.00 39.96 ATOM 1229 ND2 ASN 823 28.356 7.000 35.244 1.00 39.54 ATOM 1230 C ASN 823 25.009 4.550 32.908 1.00 35.90 ATOM 1231 O ASN 823 24.470 5.523 32.380 1.00 35.84 ATOM 1232 N GLN 824 24.410 3.371 33.026 1.00 35.27 ATOM 1233 CA GLN 824 23.064 3.128 32.512 1.00 34.89 ATOM 1234 CB GLN 824 22.544 1.759 32.968 1.00 35.03 ATOM 1235 CG GLN 824 22.418 0.736 31.853 1.00 35.35 ATOM 1236 CD GLN 824 21.877 1.332 30.573 1.00 36.02 ATOM 1237 OE1 GLN 824 20.725 1.789 30.507 1.00 36.30 ATOM 1238 NE2 GLN 824 22.713 1.341 29.538 1.00 36.24 ATOM 1239 C GLN 824 22.016 4.159 32.879 1.00 34.22 ATOM 1240 O GLN 824 21.240 4.589 32.029 1.00 34.41 ATOM 1241 N LYS 825 21.980 4.534 34.150 1.00 33.69 ATOM 1242 CA LYS 825 20.989 5.478 34.645 1.00 33.20 ATOM 1243 CB LYS 825 21.240 5.765 36.120 1.00 33.85 ATOM 1244 CG LYS 825 20.044 6.323 36.860 1.00 34.71 ATOM 1245 CD LYS 825 18.931 5.304 36.990 1.00 35.57 ATOM 1246 CE LYS 825 17.744 5.903 37.732 1.00 36.43 ATOM 1247 NZ LYS 825 18.151 6.447 39.063 1.00 37.01 ATOM 1248 C LYS 825 20.929 6.788 33.870 1.00 32.38 ATOM 1249 O LYS 825 19.857 7.329 33.656 1.00 32.60 ATOM 1250 N PHE 826 22.077 7.299 33.453 1.00 31.31 ATOM 1251 CA PHE 826 22.094 8.543 32.709 1.00 30.15 ATOM 1252 CB PHE 826 23.496 9.152 32.709 1.00 30.44 ATOM 1253 CG PHE 826 24.003 9.506 34.076 1.00 31.16 ATOM 1254 CD1 PHE 826 25.088 10.364 34.226 1.00 32.02 ATOM 1255 CD2 PHE 826 23.415 8.973 35.219 1.00 31.65 ATOM 1256 CE1 PHE 826 25.579 10.682 35.496 1.00 31.86 ATOM 1257 CE2 PHE 826 23.900 9.285 36.490 1.00 31.49 ATOM 1258 CZ PHE 826 24.983 10.140 36.624 1.00 31.58 ATOM 1259 C PHE 826 21.618 8.307 31.283 1.00 28.87 ATOM 1260 O PHE 826 20.921 9.142 30.720 1.00 28.43 ATOM 1261 N PHE 827 21.989 7.166 30.708 1.00 27.44 ATOM 1262 CA PHE 827 21.568 6.833 29.354 1.00 26.53 ATOM 1263 CB PHE 827 22.122 5.476 28.935 1.00 25.44 ATOM 1264 CG PHE 827 21.708 5.061 27.556 1.00 24.15 ATOM 1265 CD1 PHE 827 22.592 5.157 26.493 1.00 23.63 ATOM 1266 CD2 PHE 827 20.425 4.599 27.312 1.00 23.97 ATOM 1267 CE1 PHE 827 22.202 4.800 25.203 1.00 23.50 ATOM 1268 CE2 PHE 827 20.026 4.241 26.016 1.00 23.68 ATOM 1269 CZ PHE 827 20.913 4.342 24.968 1.00 22.72 ATOM 1270 C PHE 827 20.045 6.788 29.340 1.00 26.44 ATOM 1271 O PHE 827 19.406 7.407 28.492 1.00 26.04 ATOM 1272 N ASP 828 19.475 6.052 30.296 1.00 26.33 ATOM 1273 CA ASP 828 18.022 5.931 30.436 1.00 26.22 ATOM 1274 CB ASP 828 17.669 5.082 31.654 1.00 27.34 ATOM 1275 CG ASP 828 18.195 3.673 31.567 1.00 28.47 ATOM 1276 OD1 ASP 828 18.511 3.119 32.644 1.00 29.17 ATOM 1277 OD2 ASP 828 18.281 3.117 30.448 1.00 28.69 ATOM 1278 C ASP 828 17.332 7.288 30.601 1.00 25.75 ATOM 1279 O ASP 828 16.266 7.506 30.052 1.00 25.47 ATOM 1280 N GLU 829 17.915 8.191 31.382 1.00 25.59 ATOM 1281 CA GLU 829 17.292 9.494 31.559 1.00 26.44 ATOM 1282 CB GLU 829 18.006 10.303 32.650 1.00 28.25 ATOM 1283 CG GLU 829 17.415 11.696 32.898 1.00 30.25 ATOM 1284 CD GLU 829 18.210 12.499 33.925 1.00 32.25 ATOM 1285 OE1 GLU 829 19.432 12.719 33.717 1.00 33.52 ATOM 1286 OE2 GLU 829 17.618 12.918 34.946 1.00 33.33 ATOM 1287 C GLU 829 17.343 10.233 30.223 1.00 25.94 ATOM 1288 O GLU 829 16.342 10.797 29.777 1.00 25.19 ATOM 1289 N LEU 830 18.506 10.208 29.575 1.00 25.30 ATOM 1290 CA LEU 830 18.645 10.859 28.282 1.00 25.23 ATOM 1291 CB LEU 830 20.072 10.736 27.761 1.00 26.02 ATOM 1292 CG LEU 830 20.940 11.959 28.038 1.00 27.07 ATOM 1293 CD1 LEU 830 21.096 12.139 29.555 1.00 28.25 ATOM 1294 CD2 LEU 830 22.298 11.793 27.362 1.00 27.32 ATOM 1295 C LEU 830 17.678 10.249 27.279 1.00 25.12 ATOM 1296 O LEU 830 16.943 10.966 26.606 1.00 24.67 ATOM 1297 N ARG 831 17.668 8.922 27.188 1.00 25.07 ATOM 1298 CA ARG 831 16.768 8.253 26.261 1.00 25.48 ATOM 1299 CB ARG 831 16.913 6.732 26.358 1.00 25.48 ATOM 1300 CG ARG 831 15.953 5.993 25.441 1.00 25.24 ATOM 1301 CD ARG 831 16.261 4.499 25.357 1.00 25.58 ATOM 1302 NE ARG 831 15.158 3.781 24.719 1.00 25.78 ATOM 1303 CZ ARG 831 14.822 3.879 23.436 1.00 25.10 ATOM 1304 NH1 ARG 831 15.507 4.661 22.617 1.00 24.95 ATOM 1305 NH2 ARG 831 13.774 3.213 22.982 1.00 25.49 ATOM 1306 C ARG 831 15.319 8.654 26.526 1.00 25.51 ATOM 1307 O ARG 831 14.559 8.929 25.590 1.00 25.14 ATOM 1308 N MET 832 14.948 8.697 27.804 1.00 25.71 ATOM 1309 CA MET 832 13.591 9.070 28.202 1.00 26.13 ATOM 1310 CB MET 832 13.393 8.855 29.706 1.00 26.07 ATOM 1311 CG MET 832 12.280 9.683 30.316 1.00 25.80 ATOM 1312 SD MET 832 11.876 9.192 32.002 1.00 28.43 ATOM 1313 CE MET 832 12.867 10.272 32.995 1.00 26.55 ATOM 1314 C MET 832 13.255 10.518 27.847 1.00 25.99 ATOM 1315 O MET 832 12.114 10.828 27.518 1.00 26.12 ATOM 1316 N ASN 833 14.241 11.403 27.916 1.00 26.07 ATOM 1317 CA ASN 833 13.996 12.800 27.590 1.00 26.18 ATOM 1318 CB ASN 833 15.129 13.668 28.126 1.00 26.83 ATOM 1319 CG ASN 833 15.026 13.874 29.625 1.00 27.86 ATOM 1320 OD1 ASN 833 16.004 14.206 30.297 1.00 28.97 ATOM 1321 ND2 ASN 833 13.827 13.682 30.156 1.00 27.93 ATOM 1322 C ASN 833 13.795 13.021 26.099 1.00 26.03 ATOM 1323 O ASN 833 12.989 13.856 25.701 1.00 25.96 ATOM 1324 N TYR 834 14.513 12.267 25.277 1.00 25.89 ATOM 1325 CA TYR 834 14.375 12.385 23.835 1.00 26.53 ATOM 1326 CB TYR 834 15.572 11.732 23.142 1.00 25.95 ATOM 1327 CG TYR 834 16.759 12.670 23.114 1.00 25.75 ATOM 1328 CD1 TYR 834 17.008 13.471 22.004 1.00 25.23 ATOM 1329 CE1 TYR 834 18.028 14.418 22.017 1.00 25.38 ATOM 1330 CD2 TYR 834 17.566 12.839 24.239 1.00 25.31 ATOM 1331 CE2 TYR 834 18.584 13.784 24.260 1.00 24.66 ATOM 1332 CZ TYR 834 18.813 14.569 23.149 1.00 24.63 ATOM 1333 OH TYR 834 19.831 15.496 23.153 1.00 24.47 ATOM 1334 C TYR 834 13.054 11.784 23.364 1.00 27.63 ATOM 1335 O TYR 834 12.385 12.338 22.493 1.00 27.51 ATOM 1336 N ILE 835 12.668 10.655 23.948 1.00 28.57 ATOM 1337 CA ILE 835 11.404 10.044 23.586 1.00 29.73 ATOM 1338 CB ILE 835 11.131 8.765 24.404 1.00 29.44 ATOM 1339 CG2 ILE 835 9.640 8.458 24.415 1.00 29.00 ATOM 1340 CG1 ILE 835 11.940 7.598 23.828 1.00 29.23 ATOM 1341 CD1 ILE 835 11.798 6.295 24.604 1.00 28.37 ATOM 1342 C ILE 835 10.294 11.053 23.861 1.00 31.03 ATOM 1343 O ILE 835 9.354 11.171 23.084 1.00 30.89 ATOM 1344 N LYS 836 10.410 11.777 24.971 1.00 32.73 ATOM 1345 CA LYS 836 9.411 12.772 25.335 1.00 34.47 ATOM 1346 CB LYS 836 9.613 13.246 26.781 1.00 35.12 ATOM 1347 CG LYS 836 9.220 12.210 27.845 1.00 36.26 ATOM 1348 CD LYS 836 9.047 12.853 29.226 1.00 36.96 ATOM 1349 CE LYS 836 8.689 11.822 30.292 1.00 37.93 ATOM 1350 NZ LYS 836 7.331 11.205 30.106 1.00 38.44 ATOM 1351 C LYS 836 9.411 13.974 24.401 1.00 35.36 ATOM 1352 O LYS 836 8.401 14.663 24.288 1.00 35.57 ATOM 1353 N GLU 837 10.533 14.232 23.730 1.00 36.74 ATOM 1354 CA GLU 837 10.605 15.360 22.801 1.00 37.88 ATOM 1355 CB GLU 837 12.056 15.750 22.516 1.00 38.02 ATOM 1356 CG GLU 837 12.811 16.300 23.720 1.00 38.61 ATOM 1357 CD GLU 837 12.171 17.552 24.298 1.00 38.88 ATOM 1358 OE1 GLU 837 12.694 18.080 25.301 1.00 38.97 ATOM 1359 OE2 GLU 837 11.145 18.011 23.751 1.00 39.85 ATOM 1360 C GLU 837 9.902 14.995 21.502 1.00 39.04 ATOM 1361 O GLU 837 9.248 15.837 20.883 1.00 39.39 ATOM 1362 N LEU 838 10.035 13.737 21.089 1.00 40.19 ATOM 1363 CA LEU 838 9.377 13.283 19.873 1.00 41.66 ATOM 1364 CB LEU 838 9.745 11.840 19.551 1.00 40.92 ATOM 1365 CG LEU 838 9.059 11.325 18.283 1.00 40.71 ATOM 1366 CD1 LEU 838 9.337 12.287 17.131 1.00 40.13 ATOM 1367 CD2 LEU 838 9.549 9.921 17.961 1.00 40.28 ATOM 1368 C LEU 838 7.882 13.376 20.098 1.00 43.18 ATOM 1369 O LEU 838 7.117 13.684 19.187 1.00 43.33 ATOM 1370 N ASP 839 7.469 13.105 21.329 1.00 45.15 ATOM 1371 CA ASP 839 6.063 13.176 21.679 1.00 47.48 ATOM 1372 CB ASP 839 5.852 12.690 23.109 1.00 47.71 ATOM 1373 CG ASP 839 4.409 12.777 23.538 1.00 48.24 ATOM 1374 OD1 ASP 839 3.575 12.016 22.996 1.00 48.44 ATOM 1375 OD2 ASP 839 4.108 13.617 24.411 1.00 48.69 ATOM 1376 C ASP 839 5.565 14.617 21.539 1.00 48.74 ATOM 1377 O ASP 839 4.525 14.866 20.938 1.00 48.83 ATOM 1378 N ARG 840 6.314 15.566 22.086 1.00 50.51 ATOM 1379 CA ARG 840 5.922 16.965 21.999 1.00 52.38 ATOM 1380 CB ARG 840 6.739 17.805 22.985 1.00 52.53 ATOM 1381 CG ARG 840 6.330 17.586 24.444 1.00 53.33 ATOM 1382 CD ARG 840 7.033 18.548 25.395 1.00 53.67 ATOM 1383 NE ARG 840 8.378 18.110 25.759 1.00 53.99 ATOM 1384 CZ ARG 840 9.318 18.922 26.232 1.00 54.20 ATOM 1385 NH1 ARG 840 9.057 20.214 26.388 1.00 54.27 ATOM 1386 NH2 ARG 840 10.513 18.447 26.558 1.00 54.13 ATOM 1387 C ARG 840 6.041 17.534 20.585 1.00 53.55 ATOM 1388 O ARG 840 5.293 18.436 20.211 1.00 53.71 ATOM 1389 N ILE 841 6.979 17.021 19.796 1.00 54.98 ATOM 1390 CA ILE 841 7.120 17.511 18.433 1.00 56.52 ATOM 1391 CB ILE 841 8.440 17.040 17.784 1.00 56.31 ATOM 1392 CG2 ILE 841 8.442 15.540 17.629 1.00 56.61 ATOM 1393 CG1 ILE 841 8.600 17.678 16.405 1.00 56.35 ATOM 1394 CD1 ILE 841 8.600 19.187 16.425 1.00 56.59 ATOM 1395 C ILE 841 5.927 16.955 17.659 1.00 57.71 ATOM 1396 O ILE 841 5.593 17.414 16.564 1.00 57.95 ATOM 1397 N ILE 842 5.285 15.955 18.247 1.00 59.05 ATOM 1398 CA ILE 842 4.115 15.345 17.644 1.00 60.55 ATOM 1399 CB ILE 842 3.929 13.899 18.145 1.00 60.34 ATOM 1400 CG2 ILE 842 2.539 13.386 17.786 1.00 60.36 ATOM 1401 CG1 ILE 842 5.014 13.002 17.552 1.00 60.28 ATOM 1402 CD1 ILE 842 4.968 12.899 16.046 1.00 60.36 ATOM 1403 C ILE 842 2.886 16.173 18.021 1.00 61.84 ATOM 1404 O ILE 842 2.077 16.521 17.165 1.00 62.01 ATOM 1405 N ALA 843 2.768 16.503 19.304 1.00 63.28 ATOM 1406 CA ALA 843 1.636 17.274 19.811 1.00 64.93 ATOM 1407 CB ALA 843 1.202 16.719 21.168 1.00 64.96 ATOM 1408 C ALA 843 1.921 18.770 19.931 1.00 66.08 ATOM 1409 O ALA 843 2.657 19.346 19.125 1.00 66.22 ATOM 1410 N CYS 844 1.327 19.393 20.946 1.00 67.46 ATOM 1411 CA CYS 844 1.499 20.825 21.187 1.00 68.93 ATOM 1412 CB CYS 844 0.818 21.235 22.496 1.00 69.02 ATOM 1413 SG CYS 844 1.052 22.983 22.913 1.00 69.96 ATOM 1414 C CYS 844 2.965 21.264 21.224 1.00 69.69 ATOM 1415 O CYS 844 3.734 20.858 22.103 1.00 69.75 ATOM 1416 N ALA 845 3.331 22.104 20.260 1.00 70.51 ATOM 1417 CA ALA 845 4.685 22.631 20.138 1.00 71.20 ATOM 1418 CB ALA 845 5.697 21.488 20.150 1.00 71.12 ATOM 1419 C ALA 845 4.805 23.418 18.837 1.00 71.67 ATOM 1420 O ALA 845 5.173 24.599 18.838 1.00 71.61 ATOM 1421 N ALA 846 4.473 22.754 17.732 1.00 72.18 ATOM 1422 CA ALA 846 4.555 23.359 16.411 1.00 72.76 ATOM 1423 CB ALA 846 5.743 22.785 15.670 1.00 72.64 ATOM 1424 C ALA 846 3.291 23.178 15.572 1.00 73.29 ATOM 1425 O ALA 846 2.639 24.157 15.204 1.00 73.45 ATOM 1426 N LYS 847 2.949 21.925 15.273 1.00 73.74 ATOM 1427 CA LYS 847 1.780 21.617 14.450 1.00 74.06 ATOM 1428 CB LYS 847 1.558 20.100 14.383 1.00 74.11 ATOM 1429 CG LYS 847 2.498 19.384 13.412 1.00 74.21 ATOM 1430 CD LYS 847 2.359 19.953 12.002 1.00 74.15 ATOM 1431 CE LYS 847 3.331 19.313 11.026 1.00 74.04 ATOM 1432 NZ LYS 847 3.206 19.907 9.662 1.00 73.48 ATOM 1433 C LYS 847 0.478 22.307 14.842 1.00 74.24 ATOM 1434 O LYS 847 0.203 22.539 16.021 1.00 74.15 ATOM 1435 N ALA 848 −0.311 22.632 13.820 1.00 74.54 ATOM 1436 CA ALA 848 −1.600 23.296 13.981 1.00 74.71 ATOM 1437 CB ALA 848 −1.786 24.350 12.885 1.00 74.65 ATOM 1438 C ALA 848 −2.755 22.289 13.948 1.00 74.78 ATOM 1439 O ALA 848 −3.691 22.386 14.746 1.00 75.01 ATOM 1440 N PRO 849 −2.715 21.318 13.016 1.00 74.70 ATOM 1441 CD PRO 849 −1.791 21.178 11.874 1.00 74.87 ATOM 1442 CA PRO 849 −3.789 20.320 12.936 1.00 74.53 ATOM 1443 CB PRO 849 −3.627 19.756 11.529 1.00 74.63 ATOM 1444 CG PRO 849 −2.143 19.803 11.336 1.00 74.79 ATOM 1445 C PRO 849 −3.632 19.250 14.014 1.00 74.18 ATOM 1446 O PRO 849 −2.519 18.800 14.286 1.00 74.25 ATOM 1447 N THR 850 −4.746 18.848 14.621 1.00 73.78 ATOM 1448 CA THR 850 −4.736 17.840 15.682 1.00 73.31 ATOM 1449 CB THR 850 −6.180 17.441 16.067 1.00 73.45 ATOM 1450 OG1 THR 850 −6.144 16.387 17.038 1.00 73.57 ATOM 1451 CG2 THR 850 −6.961 16.990 14.832 1.00 73.61 ATOM 1452 C THR 850 −3.933 16.578 15.329 1.00 72.80 ATOM 1453 O THR 850 −4.467 15.613 14.771 1.00 72.81 ATOM 1454 N SER 851 −2.648 16.591 15.675 1.00 71.96 ATOM 1455 CA SER 851 −1.751 15.471 15.393 1.00 71.07 ATOM 1456 CB SER 851 −0.664 15.924 14.405 1.00 71.04 ATOM 1457 OG SER 851 0.243 14.881 14.097 1.00 70.59 ATOM 1458 C SER 851 −1.099 14.932 16.670 1.00 70.34 ATOM 1459 O SER 851 −0.015 15.370 17.047 1.00 70.40 ATOM 1460 N CYS 852 −1.759 13.982 17.333 1.00 69.35 ATOM 1461 CA CYS 852 −1.225 13.395 18.565 1.00 68.12 ATOM 1462 CB CYS 852 −1.375 14.384 19.735 1.00 68.59 ATOM 1463 SG CYS 852 −3.043 15.096 19.978 1.00 69.59 ATOM 1464 C CYS 852 −1.854 12.050 18.944 1.00 66.82 ATOM 1465 O CYS 852 −1.848 11.662 20.113 1.00 66.81 ATOM 1466 N SER 853 −2.382 11.334 17.956 1.00 65.27 ATOM 1467 CA SER 853 −3.007 10.041 18.211 1.00 63.60 ATOM 1468 CB SER 853 −4.373 9.970 17.514 1.00 63.93 ATOM 1469 OG SER 853 −4.272 10.324 16.148 1.00 63.97 ATOM 1470 C SER 853 −2.134 8.862 17.778 1.00 62.15 ATOM 1471 O SER 853 −1.316 8.371 18.562 1.00 62.22 ATOM 1472 N ARG 854 −2.305 8.411 16.537 1.00 60.15 ATOM 1473 CA ARG 854 −1.532 7.282 16.021 1.00 58.00 ATOM 1474 CB ARG 854 −2.376 6.455 15.035 1.00 58.75 ATOM 1475 CG ARG 854 −2.622 7.112 13.680 1.00 59.49 ATOM 1476 CD ARG 854 −3.460 6.203 12.778 1.00 60.59 ATOM 1477 NE ARG 854 −3.671 6.744 11.429 1.00 61.32 ATOM 1478 CZ ARG 854 −4.324 7.873 11.150 1.00 61.65 ATOM 1479 NH1 ARG 854 −4.848 8.611 12.124 1.00 61.58 ATOM 1480 NH2 ARG 854 −4.454 8.267 9.887 1.00 61.67 ATOM 1481 C ARG 854 −0.241 7.730 15.339 1.00 55.94 ATOM 1482 O ARG 854 0.411 6.952 14.653 1.00 55.60 ATOM 1483 N ARG 855 0.129 8.987 15.540 1.00 53.73 ATOM 1484 CA ARG 855 1.342 9.528 14.946 1.00 51.51 ATOM 1485 CB ARG 855 1.337 11.051 15.063 1.00 50.74 ATOM 1486 CG ARG 855 2.404 11.739 14.247 1.00 49.65 ATOM 1487 CD ARG 855 2.238 11.446 12.760 1.00 48.66 ATOM 1488 NE ARG 855 3.212 12.183 11.966 1.00 46.95 ATOM 1489 CZ ARG 855 3.327 12.088 10.650 1.00 46.55 ATOM 1490 NH1 ARG 855 2.526 11.280 9.967 1.00 45.80 ATOM 1491 NH2 ARG 855 4.248 12.802 10.020 1.00 46.32 ATOM 1492 C ARG 855 2.580 8.955 15.642 1.00 50.40 ATOM 1493 O ARG 855 3.633 8.777 15.026 1.00 50.15 ATOM 1494 N PHE 856 2.448 8.669 16.933 1.00 48.68 ATOM 1495 CA PHE 856 3.554 8.117 17.694 1.00 46.90 ATOM 1496 CB PHE 856 3.327 8.325 19.196 1.00 47.21 ATOM 1497 CG PHE 856 4.461 7.836 20.058 1.00 47.24 ATOM 1498 CD1 PHE 856 5.710 8.448 20.004 1.00 47.28 ATOM 1499 CD2 PHE 856 4.278 6.766 20.930 1.00 47.12 ATOM 1500 CE1 PHE 856 6.761 8.000 20.811 1.00 47.48 ATOM 1501 CE2 PHE 856 5.324 6.312 21.743 1.00 47.30 ATOM 1502 CZ PHE 856 6.566 6.929 21.684 1.00 47.08 ATOM 1503 C PHE 856 3.705 6.633 17.393 1.00 45.50 ATOM 1504 O PHE 856 4.825 6.122 17.331 1.00 45.41 ATOM 1505 N TYR 857 2.584 5.940 17.196 1.00 43.60 ATOM 1506 CA TYR 857 2.643 4.511 16.910 1.00 41.70 ATOM 1507 CB TYR 857 1.249 3.880 16.893 1.00 42.01 ATOM 1508 CG TYR 857 1.271 2.393 16.574 1.00 42.12 ATOM 1509 CD1 TYR 857 1.713 1.462 17.512 1.00 42.35 ATOM 1510 CE1 TYR 857 1.785 0.100 17.206 1.00 42.40 ATOM 1511 CD2 TYR 857 0.895 1.923 15.315 1.00 42.37 ATOM 1512 CE2 TYR 857 0.967 0.562 14.997 1.00 42.18 ATOM 1513 CZ TYR 857 1.414 −0.341 15.946 1.00 42.56 ATOM 1514 OH TYR 857 1.516 −1.681 15.629 1.00 42.93 ATOM 1515 C TYR 857 3.323 4.222 15.582 1.00 40.32 ATOM 1516 O TYR 857 4.092 3.266 15.478 1.00 40.51 ATOM 1517 N GLN 858 3.047 5.023 14.558 1.00 38.44 ATOM 1518 CA GLN 858 3.682 4.764 13.271 1.00 36.90 ATOM 1519 CB GLN 858 2.888 5.372 12.104 1.00 37.28 ATOM 1520 CG GLN 858 2.188 6.677 12.379 1.00 37.76 ATOM 1521 CD GLN 858 0.934 6.820 11.536 1.00 38.54 ATOM 1522 OE1 GLN 858 0.165 5.862 11.391 1.00 38.69 ATOM 1523 NE2 GLN 858 0.712 8.012 10.982 1.00 38.73 ATOM 1524 C GLN 858 5.134 5.201 13.210 1.00 35.37 ATOM 1525 O GLN 858 5.949 4.501 12.612 1.00 35.14 ATOM 1526 N LEU 859 5.471 6.330 13.834 1.00 33.65 ATOM 1527 CA LEU 859 6.859 6.782 13.826 1.00 32.33 ATOM 1528 CB LEU 859 6.995 8.191 14.412 1.00 32.33 ATOM 1529 CC LEU 859 6.425 9.362 13.596 1.00 32.70 ATOM 1530 CD1 LEU 859 6.736 10.688 14.294 1.00 32.82 ATOM 1531 CD2 LEU 859 7.017 9.368 12.210 1.00 32.87 ATOM 1532 C LEU 859 7.743 5.806 14.607 1.00 31.28 ATOM 1533 O LEU 859 8.844 5.475 14.174 1.00 30.56 ATOM 1534 N THR 860 7.264 5.339 15.754 1.00 30.60 ATOM 1535 CA THR 860 8.043 4.389 16.550 1.00 30.04 ATOM 1536 CB THR 860 7.398 4.142 17.938 1.00 30.12 ATOM 1537 OG1 THR 860 6.024 3.776 17.777 1.00 30.74 ATOM 1538 CG2 THR 860 7.472 5.403 18.780 1.00 29.71 ATOM 1539 C THR 860 8.162 3.083 15.764 1.00 29.10 ATOM 1540 O THR 860 9.114 2.329 15.926 1.00 28.88 ATOM 1541 N LYS 861 7.191 2.839 14.893 1.00 28.36 ATOM 1542 CA LYS 861 7.202 1.659 14.039 1.00 27.61 ATOM 1543 CB LYS 861 5.855 1.517 13.334 1.00 28.44 ATOM 1544 CG LYS 861 5.025 0.365 13.819 1.00 29.58 ATOM 1545 CD LYS 861 5.650 −0.948 13.420 1.00 30.16 ATOM 1546 CE LYS 861 4.869 −2.106 13.998 1.00 30.39 ATOM 1547 NZ LYS 861 5.567 −3.402 13.771 1.00 31.31 ATOM 1548 C LYS 861 8.291 1.885 12.991 1.00 26.55 ATOM 1549 O LYS 861 9.097 1.002 12.680 1.00 26.06 ATOM 1550 N LEU 862 8.300 3.088 12.439 1.00 25.35 ATOM 1551 CA LEU 862 9.288 3.421 11.439 1.00 24.93 ATOM 1552 CB LEU 862 9.030 4.818 10.891 1.00 24.31 ATOM 1553 CG LEU 862 10.060 5.242 9.855 1.00 24.36 ATOM 1554 CD1 LEU 862 9.948 4.337 8.645 1.00 23.62 ATOM 1555 CD2 LEU 862 9.846 6.687 9.484 1.00 23.25 ATOM 1556 C LEU 862 10.692 3.332 12.038 1.00 24.54 ATOM 1557 O LEU 862 11.581 2.748 11.433 1.00 24.21 ATOM 1558 N LEU 863 10.894 3.901 13.227 1.00 24.64 ATOM 1559 CA LEU 863 12.216 3.843 13.868 1.00 24.87 ATOM 1560 CB LEU 863 12.216 4.613 15.188 1.00 24.73 ATOM 1561 CG LEU 863 12.307 6.129 15.049 1.00 25.00 ATOM 1562 CD1 LEU 863 12.356 6.762 16.428 1.00 24.84 ATOM 1563 CD2 LEU 863 13.554 6.483 14.240 1.00 25.12 ATOM 1564 C LEU 863 12.689 2.402 14.105 1.00 24.37 ATOM 1565 O LEU 863 13.837 2.068 13.826 1.00 24.46 ATOM 1566 N ASP 864 11.815 1.549 14.622 1.00 24.20 ATOM 1567 CA ASP 864 12.190 0.154 14.835 1.00 24.33 ATOM 1568 CB ASP 864 11.014 −0.626 15.438 1.00 25.03 ATOM 1569 CG ASP 864 10.670 −0.189 16.878 1.00 26.02 ATOM 1570 OD1 ASP 864 11.490 0.519 17.518 1.00 25.80 ATOM 1571 OD2 ASP 864 9.578 −0.573 17.367 1.00 25.23 ATOM 1572 C ASP 864 12.598 −0.511 13.499 1.00 24.21 ATOM 1573 O ASP 864 13.473 −1.383 13.460 1.00 24.29 ATOM 1574 N SER 865 11.966 −0.090 12.404 1.00 23.73 ATOM 1575 CA SER 865 12.237 −0.682 11.098 1.00 23.19 ATOM 1576 CB SER 865 11.217 −0.194 10.052 1.00 22.65 ATOM 1577 OG SER 865 11.527 1.099 9.565 1.00 21.98 ATOM 1578 C SER 865 13.659 −0.432 10.611 1.00 23.12 ATOM 1579 O SER 865 14.149 −1.138 9.738 1.00 23.62 ATOM 1580 N VAL 866 14.331 0.562 11.178 1.00 22.48 ATOM 1581 CA VAL 866 15.708 0.839 10.786 1.00 21.73 ATOM 1582 CB VAL 866 16.190 2.188 11.345 1.00 20.85 ATOM 1583 CG1 VAL 866 17.665 2.377 11.012 1.00 20.74 ATOM 1584 CG2 VAL 866 15.354 3.317 10.784 1.00 19.44 ATOM 1585 C VAL 866 16.653 −0.236 11.338 1.00 21.81 ATOM 1586 O VAL 866 17.656 −0.598 10.723 1.00 22.06 ATOM 1587 N GLN 867 16.327 −0.744 12.512 1.00 21.74 ATOM 1588 CA GLN 867 17.178 −1.722 13.153 1.00 21.73 ATOM 1589 CB GLN 867 16.607 −2.055 14.527 1.00 22.18 ATOM 1590 CG GLN 867 16.498 −0.829 15.412 1.00 22.59 ATOM 1591 CD GLN 867 17.837 −0.158 15.638 1.00 22.57 ATOM 1592 OE1 GLN 867 18.826 −0.819 15.953 1.00 23.51 ATOM 1593 NE2 GLN 867 17.873 1.158 15.497 1.00 22.55 ATOM 1594 C GLN 867 17.481 −2.994 12.372 1.00 21.37 ATOM 1595 O GLN 867 18.650 −3.330 12.187 1.00 21.44 ATOM 1596 N PRO 868 16.448 −3.733 11.923 1.00 21.04 ATOM 1597 CD PRO 868 14.989 −3.550 12.037 1.00 21.82 ATOM 1598 CA PRO 868 16.770 −4.948 11.175 1.00 20.68 ATOM 1599 CB PRO 868 15.390 −5.545 10.841 1.00 20.96 ATOM 1600 CG PRO 868 14.468 −4.383 10.884 1.00 21.59 ATOM 1601 C PRO 868 17.617 −4.622 9.950 1.00 20.19 ATOM 1602 O PRO 868 18.479 −5.403 9.551 1.00 20.47 ATOM 1603 N ILE 869 17.383 −3.455 9.362 1.00 19.77 ATOM 1604 CA ILE 869 18.175 −3.037 8.216 1.00 18.93 ATOM 1605 CB ILE 869 17.656 −1.698 7.649 1.00 18.67 ATOM 1606 CG2 ILE 869 18.605 −1.185 6.545 1.00 18.45 ATOM 1607 CG1 ILE 869 16.235 −1.890 7.121 1.00 18.20 ATOM 1608 CD1 ILE 869 15.597 −0.642 6.543 1.00 17.92 ATOM 1609 C ILE 869 19.641 −2.881 8.665 1.00 18.84 ATOM 1610 O ILE 869 20.551 −3.408 8.023 1.00 17.81 ATOM 1611 N ALA 870 19.863 −2.169 9.773 1.00 18.50 ATOM 1612 CA ALA 870 21.221 −1.964 10.280 1.00 19.01 ATOM 1613 CB ALA 870 21.207 −1.118 11.563 1.00 17.32 ATOM 1614 C ALA 870 21.898 −3.305 10.541 1.00 19.42 ATOM 1615 O ALA 870 23.104 −3.452 10.329 1.00 19.55 ATOM 1616 N ARG 871 21.116 −4.286 10.981 1.00 20.41 ATOM 1617 CA ARG 871 21.654 −5.615 11.274 1.00 21.54 ATOM 1618 CB ARG 871 20.619 −6.462 12.038 1.00 21.89 ATOM 1619 CG ARG 871 21.042 −7.895 12.307 1.00 23.32 ATOM 1620 CD ARG 871 22.427 −7.959 12.926 1.00 25.37 ATOM 1621 NE ARG 871 22.888 −9.331 13.146 1.00 26.95 ATOM 1622 CZ ARG 871 22.292 −10.205 13.953 1.00 26.98 ATOM 1623 NH1 ARG 871 21.202 −9.854 14.617 1.00 27.28 ATOM 1624 NH2 ARG 871 22.803 −11.420 14.116 1.00 26.87 ATOM 1625 C ARG 871 22.096 −6.316 9.992 1.00 22.03 ATOM 1626 O ARG 871 23.135 −6.976 9.970 1.00 22.91 ATOM 1627 N GLU 872 21.316 −6.171 8.922 1.00 21.94 ATOM 1628 CA GLU 872 21.683 −6.772 7.647 1.00 21.84 ATOM 1629 CB GLU 872 20.613 −6.515 6.578 1.00 23.42 ATOM 1630 CG GLU 872 19.305 −7.281 6.758 1.00 25.54 ATOM 1631 CD GLU 872 18.418 −7.213 5.518 1.00 27.28 ATOM 1632 OE1 GLU 872 17.294 −7.766 5.553 1.00 28.13 ATOM 1633 OE2 GLU 872 18.845 −6.612 4.503 1.00 27.83 ATOM 1634 C GLU 872 23.005 −6.198 7.161 1.00 21.22 ATOM 1635 O GLU 872 23.888 −6.941 6.742 1.00 21.80 ATOM 1636 N LEU 873 23.140 −4.873 7.222 1.00 20.38 ATOM 1637 CA LEU 873 24.353 −4.198 6.773 1.00 19.25 ATOM 1638 CB LEU 873 24.115 −2.677 6.674 1.00 19.83 ATOM 1639 CG LEU 873 23.051 −2.181 5.675 1.00 19.60 ATOM 1640 CD1 LEU 873 23.053 −0.665 5.635 1.00 20.16 ATOM 1641 CD2 LEU 873 23.330 −2.726 4.279 1.00 19.48 ATOM 1642 C LEU 873 25.530 −4.489 7.690 1.00 19.03 ATOM 1643 O LEU 873 26.670 −4.505 7.252 1.00 18.60 ATOM 1644 N HIS 874 25.264 −4.720 8.972 1.00 18.94 ATOM 1645 CA HIS 874 26.354 −5.037 9.880 1.00 18.94 ATOM 1646 CB HIS 874 25.881 −5.078 11.337 1.00 18.69 ATOM 1647 CG HIS 874 25.715 −3.729 11.956 1.00 17.78 ATOM 1648 CD2 HIS 874 26.460 −2.605 11.855 1.00 17.59 ATOM 1649 ND1 HIS 874 24.703 −3.440 12.843 1.00 17.53 ATOM 1650 CE1 HIS 874 24.835 −2.196 13.267 1.00 17.42 ATOM 1651 NE2 HIS 874 25.894 −1.667 12.685 1.00 17.13 ATOM 1652 C HIS 874 26.879 −6.396 9.482 1.00 18.68 ATOM 1653 O HIS 874 28.084 −6.611 9.418 1.00 18.92 ATOM 1654 N GLN 875 25.971 −7.313 9.202 1.00 19.13 ATOM 1655 CA GLN 875 26.374 −8.654 8.810 1.00 20.22 ATOM 1656 CB GLN 875 25.141 −9.518 8.582 1.00 20.38 ATOM 1657 CG GLN 875 25.441 −10.988 8.502 1.00 21.65 ATOM 1658 CD GLN 875 26.165 −11.480 9.742 1.00 21.80 ATOM 1659 OE1 GLN 875 27.387 −11.562 9.765 1.00 22.35 ATOM 1660 NE2 GLN 875 25.408 −11.787 10.786 1.00 21.86 ATOM 1661 C GLN 875 27.226 −8.609 7.536 1.00 20.68 ATOM 1662 O GLN 875 28.321 −9.185 7.475 1.00 20.84 ATOM 1663 N PHE 876 26.728 −7.904 6.527 1.00 21.33 ATOM 1664 CA PHE 876 27.432 −7.778 5.257 1.00 21.58 ATOM 1665 CB PHE 876 26.563 −7.003 4.258 1.00 22.76 ATOM 1666 CG PHE 876 27.324 −6.426 3.099 1.00 23.48 ATOM 1667 CD1 PHE 876 27.981 −5.211 3.220 1.00 24.12 ATOM 1668 CD2 PHE 876 27.354 −7.084 1.876 1.00 24.70 ATOM 1669 CE1 PHE 876 28.658 −4.646 2.139 1.00 25.07 ATOM 1670 CE2 PHE 876 28.028 −6.535 0.780 1.00 25.33 ATOM 1671 CZ PHE 876 28.684 −5.305 0.913 1.00 25.56 ATOM 1672 C PHE 876 28.782 −7.104 5.404 1.00 21.53 ATOM 1673 O PHE 876 29.788 −7.616 4.917 1.00 21.91 ATOM 1674 N THR 877 28.806 −5.952 6.063 1.00 21.43 ATOM 1675 CA THR 877 30.063 −5.220 6.249 1.00 22.00 ATOM 1676 CB THR 877 29.824 −3.884 7.014 1.00 20.78 ATOM 1677 CG2 THR 877 31.108 −3.079 7.227 1.00 15.00 ATOM 1678 OG1 THR 877 28.924 −3.062 6.286 1.00 15.00 ATOM 1679 C THR 877 31.088 −6.071 6.987 1.00 21.87 ATOM 1680 O THR 877 32.265 −6.025 6.661 1.00 21.92 ATOM 1681 N PHE 878 30.648 −6.834 7.984 1.00 22.33 ATOM 1682 CA PHE 878 31.568 −7.694 8.727 1.00 23.05 ATOM 1683 CB PHE 878 30.867 −8.381 9.899 1.00 22.33 ATOM 1684 CG PHE 878 31.666 −9.513 10.495 1.00 21.73 ATOM 1685 CD1 PHE 878 32.782 −9.254 11.288 1.00 21.25 ATOM 1686 CD2 PHE 878 31.317 −10.838 10.241 1.00 21.33 ATOM 1687 CE1 PHE 878 33.538 −10.301 11.822 1.00 20.86 ATOM 1688 CE2 PHE 878 32.069 −11.889 10.771 1.00 21.01 ATOM 1689 CZ PHE 878 33.181 −11.618 11.562 1.00 21.09 ATOM 1690 C PHE 878 32.092 −8.766 7.775 1.00 23.92 ATOM 1691 O PHE 878 33.307 −8.936 7.618 1.00 23.91 ATOM 1692 N ASP 879 31.166 −9.503 7.160 1.00 24.70 ATOM 1693 CA ASP 879 31.545 −10.540 6.203 1.00 25.62 ATOM 1694 CB ASP 879 30.318 −11.110 5.483 1.00 26.61 ATOM 1695 CG ASP 879 29.466 −12.011 6.373 1.00 28.26 ATOM 1696 OD1 ASP 879 29.869 −12.286 7.532 1.00 29.19 ATOM 1697 OD2 ASP 879 28.388 −12.446 5.903 1.00 28.52 ATOM 1698 C ASP 879 32.485 −9.937 5.161 1.00 25.71 ATOM 1699 O ASP 879 33.446 −10.586 4.743 1.00 25.95 ATOM 1700 N LEU 880 32.224 −8.690 4.759 1.00 25.33 ATOM 1701 CA LEU 880 33.050 −8.038 3.740 1.00 25.14 ATOM 1702 CB LEU 880 32.420 −6.706 3.288 1.00 24.49 ATOM 1703 CG LEU 880 33.171 −5.970 2.157 1.00 24.36 ATOM 1704 CD1 LEU 880 33.263 −6.890 0.941 1.00 24.34 ATOM 1705 CD2 LEU 880 32.486 −4.669 1.777 1.00 23.43 ATOM 1706 C LEU 880 34.494 −7.800 4.174 1.00 24.87 ATOM 1707 O LEU 880 35.414 −7.954 3.384 1.00 24.35 ATOM 1708 N LEU 881 34.672 −7.424 5.433 1.00 25.68 ATOM 1709 CA LEU 881 35.984 −7.151 6.010 1.00 26.42 ATOM 1710 CB LEU 881 35.823 −6.684 7.459 1.00 26.13 ATOM 1711 CG LEU 881 37.124 −6.552 8.247 1.00 25.98 ATOM 1712 CD1 LEU 881 37.921 −5.392 7.676 1.00 25.56 ATOM 1713 CD2 LEU 881 36.828 −6.335 9.726 1.00 26.07 ATOM 1714 C LEU 881 36.905 −8.358 5.988 1.00 27.40 ATOM 1715 O LEU 881 38.030 −8.284 5.498 1.00 27.41 ATOM 1716 N ILE 882 36.416 −9.466 6.532 1.00 28.64 ATOM 1717 CA ILE 882 37.187 −10.694 6.610 1.00 30.19 ATOM 1718 CB ILE 882 36.353 −11.858 7.171 1.00 29.99 ATOM 1719 CG2 ILE 882 37.279 −13.022 7.499 1.00 30.50 ATOM 1720 CG1 ILE 882 35.594 −11.420 8.431 1.00 29.82 ATOM 1721 CD1 ILE 882 36.488 −10.926 9.537 1.00 28.87 ATOM 1722 C ILE 882 37.734 −11.143 5.270 1.00 31.79 ATOM 1723 O ILE 882 38.813 −11.731 5.212 1.00 32.60 ATOM 1724 N LYS 883 36.997 −10.886 4.192 1.00 33.37 ATOM 1725 CA LYS 883 37.456 −11.298 2.873 1.00 35.08 ATOM 1726 CB LYS 883 36.473 −12.298 2.254 1.00 35.22 ATOM 1727 CG LYS 883 35.030 −12.122 2.676 1.00 35.39 ATOM 1728 CD LYS 883 34.263 −13.428 2.488 1.00 35.67 ATOM 1729 CE LYS 883 33.018 −13.508 3.387 1.00 35.63 ATOM 1730 NZ LYS 883 33.347 −13.507 4.849 1.00 34.49 ATOM 1731 C LYS 883 37.729 −10.165 1.901 1.00 36.23 ATOM 1732 O LYS 883 37.951 −10.409 0.720 1.00 36.16 ATOM 1733 N SER 884 37.735 −8.929 2.394 1.00 38.01 ATOM 1734 CA SER 884 38.010 −7.781 1.535 1.00 39.99 ATOM 1735 CB SER 884 37.908 −6.480 2.331 1.00 39.82 ATOM 1736 OG SER 884 38.779 −6.500 3.445 1.00 40.42 ATOM 1737 C SER 884 39.421 −7.946 0.991 1.00 41.45 ATOM 1738 O SER 884 39.857 −7.222 0.100 1.00 41.65 ATOM 1739 N HIS 885 40.129 −8.916 1.552 1.00 43.33 ATOM 1740 CA HIS 885 41.485 −9.216 1.146 1.00 45.27 ATOM 1741 CB HIS 885 42.079 −10.274 2.085 1.00 46.21 ATOM 1742 CG HIS 885 41.975 −9.918 3.539 1.00 47.51 ATOM 1743 CD2 HIS 885 42.927 −9.619 4.457 1.00 47.92 ATOM 1744 ND1 HIS 885 40.765 −9.800 4.193 1.00 47.61 ATOM 1745 CE1 HIS 885 40.977 −9.443 5.448 1.00 47.80 ATOM 1746 NE2 HIS 885 42.280 −9.326 5.635 1.00 48.03 ATOM 1747 C HIS 885 41.482 −9.732 −0.291 1.00 46.02 ATOM 1748 O HIS 885 42.195 −9.210 −1.145 1.00 46.12 ATOM 1749 N MET 886 40.650 −10.739 −0.545 1.00 46.78 ATOM 1750 CA MET 886 40.542 −11.377 −1.856 1.00 47.47 ATOM 1751 CB MET 886 40.210 −12.861 −1.660 1.00 48.40 ATOM 1752 CG MET 886 40.207 −13.696 −2.934 1.00 49.75 ATOM 1753 SD MET 886 39.580 −15.376 −2.674 1.00 51.35 ATOM 1754 CE MET 886 37.809 −15.116 −2.950 1.00 50.99 ATOM 1755 C MET 886 39.533 −10.762 −2.842 1.00 47.47 ATOM 1756 O MET 886 39.399 −11.242 −3.966 1.00 47.36 ATOM 1757 N VAL 887 38.821 −9.713 −2.441 1.00 47.33 ATOM 1758 CA VAL 887 37.851 −9.109 −3.351 1.00 46.96 ATOM 1759 CB VAL 887 36.451 −9.054 −2.723 1.00 47.00 ATOM 1760 CG1 VAL 887 35.425 −8.807 −3.799 1.00 47.21 ATOM 1761 CG2 VAL 887 36.149 −10.356 −2.003 1.00 47.16 ATOM 1762 C VAL 887 38.271 −7.702 −3.772 1.00 46.99 ATOM 1763 O VAL 887 37.553 −7.017 −4.511 1.00 46.75 ATOM 1764 N SER 888 39.435 −7.279 −3.283 1.00 46.66 ATOM 1765 CA SER 888 40.008 −5.982 −3.617 1.00 46.35 ATOM 1766 CB SER 888 40.275 −5.926 −5.123 1.00 46.40 ATOM 1767 OG SER 888 41.043 −7.042 −5.542 1.00 45.75 ATOM 1768 C SER 888 39.180 −4.765 −3.200 1.00 46.54 ATOM 1769 O SER 888 38.840 −3.922 −4.038 1.00 46.44 ATOM 1770 N VAL 889 38.876 −4.666 −1.907 1.00 46.50 ATOM 1771 CA VAL 889 38.104 −3.546 −1.375 1.00 46.35 ATOM 1772 CB VAL 889 36.750 −4.024 −0.836 1.00 46.08 ATOM 1773 CG1 VAL 889 35.952 −2.849 −0.321 1.00 46.13 ATOM 1774 CG2 VAL 889 35.986 −4.742 −1.925 1.00 46.06 ATOM 1775 C VAL 889 38.864 −2.854 −0.243 1.00 46.74 ATOM 1776 O VAL 889 39.218 −3.490 0.751 1.00 46.95 ATOM 1777 N ASP 890 39.117 −1.554 −0.391 1.00 46.98 ATOM 1778 CA ASP 890 39.834 −0.804 0.640 1.00 47.18 ATOM 1779 CB ASP 890 40.261 0.589 0.145 1.00 48.23 ATOM 1780 CG ASP 890 40.782 0.586 −1.275 1.00 49.20 ATOM 1781 OD1 ASP 890 41.642 −0.261 −1.600 1.00 50.24 ATOM 1782 OD2 ASP 890 40.339 1.452 −2.065 1.00 49.63 ATOM 1783 C ASP 890 38.946 −0.595 1.863 1.00 46.72 ATOM 1784 O ASP 890 37.725 −0.488 1.748 1.00 46.63 ATOM 1785 N PHE 891 39.568 −0.534 3.033 1.00 46.21 ATOM 1786 CA PHE 891 38.850 −0.284 4.274 1.00 45.79 ATOM 1787 CB PHE 891 38.739 −1.551 5.129 1.00 44.93 ATOM 1788 CG PHE 891 37.417 −2.260 5.003 1.00 43.95 ATOM 1789 CD1 PHE 891 37.284 −3.385 4.192 1.00 43.65 ATOM 1790 CD2 PHE 891 36.300 −1.795 5.683 1.00 43.28 ATOM 1791 CE1 PHE 891 36.060 −4.032 4.064 1.00 43.03 ATOM 1792 CE2 PHE 891 35.071 −2.437 5.561 1.00 42.87 ATOM 1793 CZ PHE 891 34.952 −3.557 4.750 1.00 42.99 ATOM 1794 C PHE 891 39.647 0.764 5.028 1.00 46.31 ATOM 1795 O PHE 891 40.804 0.535 5.376 1.00 46.19 ATOM 1796 N PRO 892 39.056 1.945 5.258 1.00 46.87 ATOM 1797 CD PRO 892 37.779 2.479 4.761 1.00 47.03 ATOM 1798 CA PRO 892 39.800 2.973 5.989 1.00 47.86 ATOM 1799 CB PRO 892 38.777 4.100 6.126 1.00 47.55 ATOM 1800 CG PRO 892 38.002 3.981 4.856 1.00 47.27 ATOM 1801 C PRO 892 40.272 2.421 7.337 1.00 48.77 ATOM 1802 O PRO 892 39.559 1.660 7.994 1.00 48.53 ATOM 1803 N GLU 893 41.478 2.804 7.737 1.00 49.95 ATOM 1804 CA GLU 893 42.069 2.330 8.987 1.00 50.96 ATOM 1805 CB GLU 893 43.278 3.194 9.349 1.00 51.80 ATOM 1806 CG GLU 893 44.243 2.521 10.314 1.00 53.18 ATOM 1807 CD GLU 893 44.688 1.145 9.833 1.00 53.98 ATOM 1808 OE1 GLU 893 43.850 0.215 9.823 1.00 54.56 ATOM 1809 OE2 GLU 893 45.874 0.995 9.461 1.00 54.43 ATOM 1810 C GLU 893 41.108 2.267 10.177 1.00 50.91 ATOM 1811 O GLU 893 41.006 1.232 10.834 1.00 51.08 ATOM 1812 N MET 894 40.415 3.368 10.454 1.00 50.86 ATOM 1813 CA MET 894 39.468 3.418 11.567 1.00 50.94 ATOM 1814 CB MET 894 38.942 4.846 11.768 1.00 51.97 ATOM 1815 CG MET 894 39.769 5.723 12.688 1.00 53.43 ATOM 1816 SD MET 894 39.094 7.410 12.777 1.00 55.12 ATOM 1817 CE MET 894 40.330 8.326 11.783 1.00 54.74 ATOM 1818 C MET 894 38.278 2.495 11.337 1.00 50.28 ATOM 1819 O MET 894 37.617 2.069 12.287 1.00 50.13 ATOM 1820 N MET 895 38.007 2.204 10.068 1.00 49.44 ATOM 1821 CA MET 895 36.885 1.351 9.682 1.00 48.35 ATOM 1822 CB MET 895 36.715 1.388 8.155 1.00 49.15 ATOM 1823 CG MET 895 35.410 0.824 7.630 1.00 49.68 ATOM 1824 SD MET 895 34.001 1.826 8.121 1.00 51.13 ATOM 1825 CE MET 895 34.136 3.188 6.979 1.00 50.63 ATOM 1826 C MET 895 37.094 −0.089 10.152 1.00 46.87 ATOM 1827 O MET 895 36.336 −0.597 10.975 1.00 46.59 ATOM 1828 N ALA 896 38.136 −0.729 9.634 1.00 45.22 ATOM 1829 CA ALA 896 38.442 −2.113 9.977 1.00 43.83 ATOM 1830 CB ALA 896 39.706 −2.563 9.247 1.00 43.37 ATOM 1831 C ALA 896 38.601 −2.324 11.481 1.00 42.67 ATOM 1832 O ALA 896 38.473 −3.440 11.976 1.00 42.42 ATOM 1833 N GLU 897 38.872 −1.243 12.200 1.00 41.32 ATOM 1834 CA GLU 897 39.058 −1.306 13.640 1.00 39.88 ATOM 1835 CB GLU 897 39.828 −0.058 14.101 1.00 40.74 ATOM 1836 CG GLU 897 40.690 −0.236 15.353 1.00 41.25 ATOM 1837 CD GLU 897 41.768 0.849 15.491 1.00 41.90 ATOM 1838 OE1 GLU 897 42.665 0.918 14.619 1.00 41.86 ATOM 1839 OE2 GLU 897 41.723 1.632 16.468 1.00 42.21 ATOM 1840 C GLU 897 37.685 −1.390 14.313 1.00 38.68 ATOM 1841 O GLU 897 37.346 −2.397 14.944 1.00 38.45 ATOM 1842 N ILE 898 36.889 −0.339 14.159 1.00 36.89 ATOM 1843 CA ILE 898 35.558 −0.308 14.745 1.00 35.29 ATOM 1844 CB ILE 898 34.799 0.981 14.366 1.00 35.18 ATOM 1845 CG2 ILE 898 33.459 1.003 15.063 1.00 34.79 ATOM 1846 CG1 ILE 898 35.614 2.212 14.782 1.00 35.43 ATOM 1847 CD1 ILE 898 34.990 3.555 14.378 1.00 34.84 ATOM 1848 C ILE 898 34.728 −1.513 14.307 1.00 33.92 ATOM 1849 O ILE 898 33.926 −2.034 15.087 1.00 33.92 ATOM 1850 N ILE 899 34.912 −1.951 13.064 1.00 32.03 ATOM 1851 CA ILE 899 34.173 −3.108 12.566 1.00 30.47 ATOM 1852 CB ILE 899 34.485 −3.404 11.099 1.00 30.35 ATOM 1853 CG2 ILE 899 33.746 −4.653 10.663 1.00 29.70 ATOM 1854 CG1 ILE 899 34.090 −2.222 10.221 1.00 30.03 ATOM 1855 CD1 ILE 899 34.481 −2.407 8.775 1.00 29.09 ATOM 1856 C ILE 899 34.528 −4.370 13.343 1.00 29.56 ATOM 1857 O ILE 899 33.668 −5.007 13.934 1.00 29.56 ATOM 1858 N SER 900 35.809 −4.717 13.339 1.00 28.34 ATOM 1859 CA SER 900 36.283 −5.916 14.007 1.00 27.55 ATOM 1860 CB SER 900 37.767 −6.124 13.711 1.00 27.86 ATOM 1861 OG SER 900 38.539 −5.030 14.182 1.00 29.18 ATOM 1862 C SER 900 36.072 −5.940 15.511 1.00 26.83 ATOM 1863 O SER 900 35.911 −7.016 16.108 1.00 26.70 ATOM 1864 N VAL 901 36.066 −4.761 16.122 1.00 25.47 ATOM 1865 CA VAL 901 35.914 −4.669 17.565 1.00 24.36 ATOM 1866 CB VAL 901 36.810 −3.539 18.132 1.00 24.53 ATOM 1867 CG1 VAL 901 36.613 −3.401 19.635 1.00 24.63 ATOM 1868 CG2 VAL 901 38.261 −3.838 17.826 1.00 24.17 ATOM 1869 C VAL 901 34.489 −4.457 18.052 1.00 23.82 ATOM 1870 O VAL 901 34.056 −5.107 18.998 1.00 23.59 ATOM 1871 N GLN 902 33.754 −3.565 17.404 1.00 23.25 ATOM 1872 CA GLN 902 32.396 −3.264 17.836 1.00 23.10 ATOM 1873 CB GLN 902 32.128 −1.771 17.652 1.00 23.43 ATOM 1874 CG GLN 902 33.026 −0.856 18.468 1.00 24.40 ATOM 1875 CD GLN 902 32.724 −0.898 19.960 1.00 25.11 ATOM 1876 OE1 GLN 902 32.836 0.108 20.652 1.00 25.82 ATOM 1877 NE2 GLN 902 32.353 −2.070 20.463 1.00 26.55 ATOM 1878 C GLN 902 31.285 −4.062 17.150 1.00 22.80 ATOM 1879 O GLN 902 30.330 −4.488 17.802 1.00 22.81 ATOM 1880 N VAL 903 31.409 −4.260 15.840 1.00 21.89 ATOM 1881 CA VAL 903 30.397 −4.985 15.083 1.00 21.01 ATOM 1882 CB VAL 903 30.753 −4.987 13.568 1.00 20.43 ATOM 1883 CG1 VAL 903 29.811 −5.891 12.783 1.00 20.60 ATOM 1884 CG2 VAL 903 30.625 −3.572 13.033 1.00 19.46 ATOM 1885 C VAL 903 30.118 −6.404 15.594 1.00 20.85 ATOM 1886 O VAL 903 28.962 −6.798 15.710 1.00 19.49 ATOM 1887 N PRO 904 31.172 −7.182 15.925 1.00 21.30 ATOM 1888 CD PRO 904 32.614 −6.943 15.734 1.00 20.97 ATOM 1889 CA PRO 904 30.941 −8.545 16.421 1.00 21.34 ATOM 1890 CB PRO 904 32.356 −9.114 16.548 1.00 20.99 ATOM 1891 CG PRO 904 33.120 −8.353 15.512 1.00 21.65 ATOM 1892 C PRO 904 30.175 −8.585 17.750 1.00 22.05 ATOM 1893 O PRO 904 29.548 −9.600 18.077 1.00 22.76 ATOM 1894 N LYS 905 30.234 −7.503 18.524 1.00 21.69 ATOM 1895 CA LYS 905 29.512 −7.464 19.791 1.00 22.05 ATOM 1896 CB LYS 905 29.823 −6.183 20.577 1.00 22.91 ATOM 1897 CG LYS 905 31.236 −6.070 21.145 1.00 23.84 ATOM 1898 CD LYS 905 31.333 −4.835 22.041 1.00 24.82 ATOM 1899 CE LYS 905 32.692 −4.710 22.736 1.00 25.65 ATOM 1900 NZ LYS 905 32.716 −3.560 23.693 1.00 25.72 ATOM 1901 C LYS 905 28.023 −7.491 19.477 1.00 21.85 ATOM 1902 O LYS 905 27.208 −7.982 20.255 1.00 21.42 ATOM 1903 N ILE 906 27.675 −6.929 18.330 1.00 21.67 ATOM 1904 CA ILE 906 26.286 −6.873 17.903 1.00 21.52 ATOM 1905 CB ILE 906 26.078 −5.742 16.827 1.00 21.27 ATOM 1906 CG2 ILE 906 24.655 −5.766 16.313 1.00 20.46 ATOM 1907 CG1 ILE 906 26.442 −4.380 17.434 1.00 20.69 ATOM 1908 CD1 ILE 906 26.272 −3.193 16.517 1.00 21.16 ATOM 1909 C ILE 906 25.865 −8.226 17.331 1.00 21.33 ATOM 1910 O ILE 906 24.902 −8.827 17.800 1.00 21.55 ATOM 1911 N LEU 907 26.607 −8.715 16.342 1.00 20.88 ATOM 1912 CA LEU 907 26.274 −9.990 15.717 1.00 21.31 ATOM 1913 CB LEU 907 27.244 −10.280 14.561 1.00 19.40 ATOM 1914 CG LEU 907 27.380 −9.060 13.634 1.00 18.63 ATOM 1915 CD1 LEU 907 28.333 −9.370 12.509 1.00 17.46 ATOM 1916 CD2 LEU 907 26.008 −8.646 13.092 1.00 17.25 ATOM 1917 C LEU 907 26.225 −11.165 16.695 1.00 21.62 ATOM 1918 O LEU 907 25.360 −12.037 16.563 1.00 22.68 ATOM 1919 N SER 908 27.118 −11.177 17.685 1.00 21.63 ATOM 1920 CA SER 908 27.150 −12.266 18.664 1.00 22.10 ATOM 1921 CB SER 908 28.564 −12.433 19.238 1.00 21.84 ATOM 1922 OG SER 908 28.952 −11.279 19.949 1.00 22.32 ATOM 1923 C SER 908 26.146 −12.060 19.802 1.00 22.09 ATOM 1924 O SER 908 26.086 −12.862 20.740 1.00 22.15 ATOM 1925 N GLY 909 25.376 −10.976 19.730 1.00 22.11 ATOM 1926 CA GLY 909 24.361 −10.718 20.743 1.00 22.44 ATOM 1927 C GLY 909 24.707 −9.946 22.006 1.00 22.41 ATOM 1928 O GLY 909 23.843 −9.735 22.854 1.00 22.66 ATOM 1929 N LYS 910 25.950 −9.519 22.152 1.00 22.78 ATOM 1930 CA LYS 910 26.332 −8.773 23.344 1.00 23.71 ATOM 1931 CB LYS 910 27.838 −8.650 23.410 1.00 24.13 ATOM 1932 CG LYS 910 28.521 −9.978 23.588 1.00 25.11 ATOM 1933 CD LYS 910 30.006 −9.783 23.775 1.00 25.46 ATOM 1934 CE LYS 910 30.613 −11.053 24.288 1.00 26.49 ATOM 1935 NZ LYS 910 29.764 −11.585 25.391 1.00 27.68 ATOM 1936 C LYS 910 25.702 −7.381 23.438 1.00 24.13 ATOM 1937 O LYS 910 25.442 −6.880 24.540 1.00 23.96 ATOM 1938 N VAL 911 25.465 −6.769 22.278 1.00 24.03 ATOM 1939 CA VAL 911 24.869 −5.445 22.182 1.00 24.31 ATOM 1940 CB VAL 911 25.868 −4.413 21.588 1.00 24.16 ATOM 1941 CG1 VAL 911 26.588 −5.010 20.444 1.00 25.18 ATOM 1942 CG2 VAL 911 25.142 −3.180 21.086 1.00 24.84 ATOM 1943 C VAL 911 23.672 −5.582 21.272 1.00 24.68 ATOM 1944 O VAL 911 23.781 −6.139 20.185 1.00 25.01 ATOM 1945 N LYS 912 22.527 −5.081 21.710 1.00 24.92 ATOM 1946 CA LYS 912 21.322 −5.192 20.910 1.00 25.57 ATOM 1947 CB LYS 912 20.429 −6.297 21.470 1.00 26.08 ATOM 1948 CG LYS 912 21.049 −7.676 21.478 1.00 26.96 ATOM 1949 CD LYS 912 20.221 −8.591 22.357 1.00 28.27 ATOM 1950 CE LYS 912 20.096 −7.986 23.747 1.00 28.84 ATOM 1951 NZ LYS 912 21.426 −7.849 24.424 1.00 28.86 ATOM 1952 C LYS 912 20.516 −3.903 20.836 1.00 25.87 ATOM 1953 O LYS 912 20.561 −3.069 21.742 1.00 25.78 ATOM 1954 N PRO 913 19.763 −3.731 19.742 1.00 26.36 ATOM 1955 CD PRO 913 19.645 −4.672 18.615 1.00 26.55 ATOM 1956 CA PRO 913 18.927 −2.550 19.520 1.00 26.76 ATOM 1957 CB PRO 913 18.356 −2.777 18.118 1.00 26.68 ATOM 1958 CG PRO 913 19.296 −3.755 17.493 1.00 27.35 ATOM 1959 C PRO 913 17.808 −2.527 20.549 1.00 27.50 ATOM 1960 O PRO 913 17.435 −3.571 21.088 1.00 27.19 ATOM 1961 N ILE 914 17.288 −1.335 20.826 1.00 28.15 ATOM 1962 CA ILE 914 16.168 −1.190 21.743 1.00 28.95 ATOM 1963 CB ILE 914 16.344 0.020 22.690 1.00 29.11 ATOM 1964 CG2 ILE 914 15.077 0.212 23.525 1.00 28.84 ATOM 1965 CG1 ILE 914 17.563 −0.195 23.598 1.00 29.16 ATOM 1966 CD1 ILE 914 17.882 0.986 24.507 1.00 29.16 ATOM 1967 C ILE 914 14.955 −0.942 20.848 1.00 29.58 ATOM 1968 O ILE 914 14.896 0.069 20.148 1.00 30.48 ATOM 1969 N TYR 915 14.010 −1.879 20.844 1.00 29.76 ATOM 1970 CA TYR 915 12.791 −1.759 20.042 1.00 29.79 ATOM 1971 CB TYR 915 12.344 −3.133 19.514 1.00 29.35 ATOM 1972 CG TYR 915 13.194 −3.687 18.405 1.00 29.23 ATOM 1973 CD1 TYR 915 14.242 −4.574 18.667 1.00 29.36 ATOM 1974 CE1 TYR 915 15.055 −5.047 17.641 1.00 29.21 ATOM 1975 CD2 TYR 915 12.981 −3.289 17.092 1.00 29.29 ATOM 1976 CE2 TYR 915 13.785 −3.749 16.064 1.00 29.70 ATOM 1977 CZ TYR 915 14.818 −4.626 16.340 1.00 29.95 ATOM 1978 OH TYR 915 15.597 −5.080 15.296 1.00 31.15 ATOM 1979 C TYR 915 11.641 −1.180 20.861 1.00 29.93 ATOM 1980 O TYR 915 11.549 −1.412 22.060 1.00 30.36 ATOM 1981 N PHE 916 10.765 −0.426 20.217 1.00 29.99 ATOM 1982 CA PHE 916 9.610 0.105 20.917 1.00 30.60 ATOM 1983 CB PHE 916 9.025 1.319 20.191 1.00 30.23 ATOM 1984 CG PHE 916 9.744 2.602 20.478 1.00 30.62 ATOM 1985 CD1 PHE 916 9.659 3.192 21.731 1.00 30.55 ATOM 1986 CD2 PHE 916 10.503 3.226 19.494 1.00 30.58 ATOM 1987 CE1 PHE 916 10.315 4.381 22.000 1.00 30.34 ATOM 1988 CE2 PHE 916 11.163 4.417 19.757 1.00 30.86 ATOM 1989 CZ PHE 916 11.068 4.994 21.010 1.00 30.74 ATOM 1990 C PHE 916 8.564 −1.014 20.982 1.00 31.09 ATOM 1991 O PHE 916 7.976 −1.259 22.038 1.00 31.66 ATOM 1992 N HIS 917 8.345 −1.696 19.858 1.00 31.14 ATOM 1993 CA HIS 917 7.373 −2.782 19.802 1.00 31.46 ATOM 1994 CB HIS 917 6.375 −2.579 18.651 1.00 30.70 ATOM 1995 CG HIS 917 5.843 −1.185 18.534 1.00 29.89 ATOM 1996 CD2 HIS 917 4.695 −0.628 18.984 1.00 29.89 ATOM 1997 ND1 HIS 917 6.514 −0.188 17.863 1.00 29.93 ATOM 1998 CE1 HIS 917 5.800 0.924 17.899 1.00 29.50 ATOM 1999 NE2 HIS 917 4.691 0.683 18.574 1.00 29.71 ATOM 2000 C HIS 917 8.093 −4.099 19.590 1.00 32.03 ATOM 2001 O HIS 917 9.218 −4.116 19.102 1.00 33.01 ATOM 2002 N ALA 918 7.442 −5.201 19.949 1.00 32.62 ATOM 2003 CA ALA 918 8.025 −6.529 19.777 1.00 32.98 ATOM 2004 CB ALA 918 7.769 −7.382 21.013 1.00 33.52 ATOM 2005 C ALA 918 7.421 −7.201 18.551 1.00 33.16 ATOM 2006 O ALA 918 7.922 −7.049 17.432 1.00 33.55 

1-12. (canceled)
 13. A molecule or molecular complex comprising all or any part of the ligand binding site defined by structure coordinates of AR-LBD amino acids V685, L700, L701, S702, S703, L704, N705, E706, L707, G708, E709, Q711, A735, 1737, Q738, Y739, S740, W741, M742, G743, L744, M745, V746, F747, A748, M749, G750, R752, Y763, F764, A765, L768, F770, M780, M787, 1869, L873, H874, F876, T877, F878, M894, M895, A896, E897, I898, I899, S900, V901, Q902, V903, P904, K905, I906 and L907 according to Table A, or a mutant or homologue of said molecule or molecular complex.
 14. The molecule or molecular complex of claim 13 wherein said mutant or homologue comprises a binding pocket that has a root mean square deviation from the backbone atoms of said AR-LBD amino acids of not more than 1.5 Angstroms or 30% sequence identity with said AR-LBD amino acids.
 15. The molecule or molecular complex of claim 13 comprising all or any part of the ligand binding site defined by structure coordinates of AR-LBD amino acids N705, W741, Q711, R752, F764, T877, M895 and I898 according to Table A, or a mutant or homologue of said molecule or molecular complex.
 16. The molecule or molecular complex of claim 15 wherein said mutant or homologue comprises a binding pocket that has a root mean square deviation from the backbone atoms of said AR-LBD amino acids of not more than 1.5 Angstroms or 30% sequence identity with said AR-LBD amino acids. 17-19. (canceled)
 20. A binding site in AR-LBD for an AR modulator in which a portion of said ligand is in van der Walls contact or hydrogen bonding contact with any portion or all of residues V685, L700, L701, S702, S703, L704, N705, E706, L707, G708, E709, Q711, A735, I737, Q738, Y739, S740, W741, M742, G743, L744, M745, V746, F747, A748, M749, G750, R752, Y763, F764, A765, L768, F770, M780, M787, I869, L873, H874, F876, T877, F878, L880, L881, V889, F891, P892, E893, M894, M895, A896, E897, I898, I899, S900, V901, Q902, V903, P904, K905, I906 or L907 of AR-LBD according to Table A.
 21. The binding site according to claim 20 wherein the AR-LBD is a homologue or mutant with 25%-95% identity to residues V685, L700, L701, S702, S703, L704, N705, E706, L707, G708, E709, Q711, A735, I737, Q738, Y739, S740, W741, M742, G743, L744, M745, V746, F747, A748, M749, G750, R752, Y763, F764, A765, L768, F770, M780, M787, I869, L873, H874, F876, T877, F878, L880, L881, V889, F891, P892, E893, M894, M895, A896, E897, I898, I899, S900, V901, Q902, V903, P904, K905, I906, or L907 of AR-LBD according to Table A.
 22. A computational method of designing an androgen receptor synthetic ligand comprising: a. using a three dimensional model of a crystallized protein comprising an AR-LBD/AR-LBD ligand complex to determine at least one interacting amino acid of the AR-LBD that interacts with at least one first chemical moiety of the AR-LBD ligand; and b. selecting at least one chemical modification of said first chemical moiety to produce a second chemical moiety with a structure that either decreases or increases an interaction between said interacting amino acid and said second chemical moiety compared to said interaction between said interacting amino acid and said first chemical moiety.
 23. A method for identifying a compound that modulates androgen receptor activity, the method comprising any combination of steps of: a. modeling test compounds that fit spatially into the AR-LBD as defined by structure coordinates according to Table A, or using a three-dimensional structural model of AR-LBD, mutant AR-LBD or AR-LBD homologue or portion thereof; b. using said structure coordinates or ligand binding site as set forth in claim 20 to identify structural and chemical features; c. employing identified structural or chemical features to design or select compounds as potential SARMs; d. employing the three-dimensional structural model or the ligand binding site to design or select compounds as potential SARMs; e. synthesizing the potential SARMs; f. screening the potential SARMs in an assay characterized by binding of a test compound to the AR-LBD; and g. modifying or replacing one or more amino acids from AR-LBD selected from the group consisting of V685, L700, L701, S702, S703, L704, N705, E706, L707, G708, E709, Q711, A735, I737, Q738, Y739, S740, W741, M742, G743, L744, M745, V746, F747, A748, M749, G750, R752, Y763, F764, A765, L768, F770, M780, M787, 1869, L873, H874, F876, T877, F878, L880, L881, V889, F891, P892, E893, M894, M895, A896, E897, I898, I899, S900, V901, Q902, V903, P904, K905, I906 or L907 of AR-LBD according to Table A.
 24. (canceled) 